AU2016363025A1

AU2016363025A1 - Modified chimeric receptors and related compositions and methods

Info

Publication number: AU2016363025A1
Application number: AU2016363025A
Authority: AU
Inventors: Archana BRAHMANDAM; Robert F. Dubose; Lucas James Thompson
Original assignee: Juno Therapeutics Inc
Current assignee: Juno Therapeutics Inc
Priority date: 2015-12-03
Filing date: 2016-12-02
Publication date: 2018-06-21
Anticipated expiration: 2036-12-02
Also published as: JP7184645B2; ES2940607T3; CN108884140A; MX2018006789A; JP2023022161A; JP2019501647A; EP4212547A1; CN108884140B; CN116041538A; EP3383892A1; EP3383892B1; CA3007262A1; US20180355014A1; MA43380A; WO2017096329A1; US12139526B2; AU2016363025B2

Abstract

Provided are chimeric receptors for engineering cells for adoptive therapy, including T cells, and the genetically engineered cells. In some embodiments, the chimeric receptors, such as chimeric antigen receptors (CARs) are modified in a junction region by one or more amino acid modifications such that peptide fragments of such region exhibit a lower binding affinity for a human leukocyte antigen (HLA) and/or the region exhibits reduced immunogenicity, including following administration to a subject. In some aspects, also provided are methods and compositions for engineering and producing cells expressing such chimeric receptors, compositions containing the cells, and method for their administration to subjects. In some embodiments, features of the chimeric receptors and engineered cells containing the chimeric receptors result in methods that provide for increased or improved activity, efficacy and/or persistence.

Description

invention.

Example 1: Analysis of Transgene Product-Specific Host Immune Responses [0276] Pre- and post-treatment peripheral blood mononuclear cells (PBMC) samples were obtained from four (4) subjects with B cell malignancies treated with autologous T cells expressing a CD19-specific CAR. The CAR included an anti-CD 19 scFv derived from murine antibody, a hinge domain, a CD28 transmembrane domain, a 4-IBB intracellular signaling domain, and a CD3-zeta intracellular signaling domain. The CAR-expressing T cells also expressed a truncated EGFR (EGFRt) as a surrogate marker by transducing cells with a lentiviral vector containing a nucleic acid encoding the CAR and a nucleic acid encoding the EGFRt surrogate marker, separated by a T2A ribosome switch domain.

[0277] Pre- and post (day 42)-infusion PBMCs obtained from the subjects were assessed to detect the presence or absence of specific anti-CAR immune responses essentially as described by Berger et al. Blood. 2006 March; 107(6): 2294-2302, Berger et al. J Virol. 2001 January

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75(2): 799-808, Riddell et al. Nature Medicine. 1996 February 2(2): 216-223, Berger et al.

Blood. 2005 February 105(4): 1640-1647. Briefly, PBMCs (responders) were stimulated in vitro with autologous gamma-irradiated cells transduced with the CAR expressed by the administered cells (stimulators at a 1:1 or 2:1 responder-to-stimulator ratio). The cultures then were assessed in a chromium release assay for cytotoxicity against autologous ⁵¹Cr-labeled CAR-transduced (“CD 19 CAR”) and non-transduced (“Mock”) T cells (targets) at various effector-to-target (E/T) ratios. Following co-incubation, release of chromium was quantified and the percentage of maximum achievable lysis in each sample determined.

[0278] The results for samples derived from one exemplary patient are shown in Figure 1, which depicts the cytolytic activity of PBMCs pre-infusion and post-infusion at day 42. Whereas no cytolytic activity specific for CAR-transduced target cells was detected in any pre-infusion PBMC-derived cultures, in two of the four subjects assessed, CAR-specific lytic activity was detected in cultures derived from post-infusion PBMC samples. These results indicate that CAR-specific immune responses can develop following a single infusion of CAR-expressing T cells.

[0279] Epitope mapping was carried out to assess region(s) of the CAR recognized by the specific immune responses. Pre- and post-infusion PBMC samples were stimulated in the presence of individual pools of multiple 15-mer peptides, with sequences representing overlapping portions (11 amino acid overlap) of the entire length of an approximately 500 amino acid sequence of the CAR expressed by the administered cells. Cells were stained with antibodies to detect CD8 and CD4 surface expression and intracellular expression of cytokine. Twenty-three (23) pools were assessed, each containing ten (10) peptides each and collectively including 125 individual overlapping peptides, with each peptide represented in at least two of the pools.

[0280] This design permitted the generation of an analytic grid to assess responses specific for individual peptides, whereby a peptide present in more than one pool detected as hits in this assay was deemed a potentially immunogenic peptide hit. For the two patients in whom a CARspecific immune response had been detected, six and three peptide hits, respectively, were identified.

[0281] Individual EFISpot assays were performed using an anti-cytokine capture antibody to assess the presence or absence oi a specific immune response for each oi these individual hits (see Berger et al. (2006); Berger et al. (2001); Riddell et al. (1996); and Berger et al. (2005),

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PCT/US2016/064861 supra). The results of an exemplary assay for one patient are shown in FIG. 2. Specific immune responses against peptides with sequences within the Vh portion of the scFv of the CAR were detected in both patients assessed (including regions within the FR1, CDR1, and FR2 regions for one patient and within the FR3 for the other). For the first patient, specific immune responses also were detected against two overlapping 15-mer peptides, each containing the junction between the transmembrane domain and costimulatory domain of the CAR (labeled “fusion site” in FIG. 2). These two overlapping 15-mer peptides had the amino acid sequences AFIIFWVKRGRKKLL (SEQ ID NO: 8) and FWVKRGRKKLLYIFK (SEQ ID NO: 9), respectively. In another study following administration with a different CAR having a murine scFv, CD28 transmembrane and costimulatory domains and a CD3 zeta domain, using similar methods, an immune response also was detected for one subject against a pool containing V_Hportions of an anti-CD19 scFv and for another subject in a pool containing junction portions.

[0282] No specific immune responses were detected in the patients by this assay against peptides within other regions. For example, in this assay, no specific responses were detected against peptides having sequences within other CDRs or framework regions of the scFv, peptides within regions of costimulatory or transmembrane domain but not spanning the junction between the two, or peptides within the EGFRt or Εϋ3-ζ region of the CAR. Specific immune responses were not detected against endogenous sequences.

Example 2: In Silico Analysis of Peptides Derived from Junction Regions of a CAR for

Binding to HLA Class I and HLA Class II [0283] T cell epitope prediction tools, available from the Immune Epitope Database and analysis resource (IEDB), were used for in silico analysis to predict MHC-binding affinities and other properties related to potential immunogenicity for each of a series of overlapping peptide sequences within a portion of an exemplary CAR sequence. The portion included a spacer having an immunoglobulin-derived hinge domain, a human CD28 transmembrane domain, a human 4-IBB costimulatory domain, and a human CD3zeta signaling domain. In the portion assessed, the hinge domain was a human IgG4 hinge domain, the CD28 transmembrane domain comprised a sequence set forth in SEQ ID NO:2 and the 4-IBB costimulatory domain contained the sequence set forth in SEQ ID NO:3. This portion thus contained three junctions between different domains derived from human sequences (which junctions may have represented sites of potential immunogenicity against a CAR upon administration to a human subject): the junction between the spacer region and transmembrane domain, the junction between the transmembrane

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PCT/US2016/064861 domain and costimulatory domain, and the junction between the costimulatory domain and intracellular signaling domain (see Figures 3A and 3B).

[0284] To identify portions of the sequence that may have particular properties making them more likely to be presented to T cells, affinities for binding to 27 individual HLA class I alleles and 56 individual HLA class II alleles were predicted for overlapping peptides along the length of the portion, of 8-14 amino acids in length and of 15 amino acids in length (containing 9-mer binding core), respectively. These alleles, collectively representing HLA alleles present in greater than 99% of the worldwide population, and their approximate frequency in the United States population are listed in Tables 1A and IB.

Table 1A: HLA class I
Class I	allele	Frequency in population
1	HLA-A*01:01	12.94
2	HLA-A*02:01	42.88
3	HLA-A*02:03	0.19
4	HLA-A*02:06	1.55
5	HLA-A*03:01	13.50
6	HLA-A*ll:01	11.60
7	HLA-A*23:01	8.30
8	HLA-A*24:02	22.56
9	HLA-A*26:01	5.36
10	HLA-A*30:01	6.29
11	HLA-A*30:02	5.21
12	HLA-A*31:01	6.87
13	HLA-A*32:01	3.71
14	HLA-A*33:01	2.62
15	HLA-A*68:01	6.36
16	HLA-A*68:02	4.79
17	HLA-B*07:02	12.96
18	HLA-B*08:01	9.23
19	HLA-B*15:01	6.54
20	HLA-B*35:01	13.03
21	HLA-B*40:01	9.79
22	HLA-B *44:02	7.22
23	HLA-B *44:03	8.96
24	HLA-B*51:01	8.51
25	HLA-B*53:01	7.26
26	HLA-B*57:01	3.49
27	HLA-B*58:01	4.82

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Table IB: HLA class II
Class II	allele	Frequency in population	Class II	allele	frequency in population
1	HLA-DRB 1*01:01	13.62	29	HLA-DQA 101:02/DQB 1 05:02	21.13
2	HLA-DRB 1*15:01	22.86	30	HLA-DQA1 01:02/DQB 1 06:02	30.74
3	HLA-DRB 1*03:01	21.82	31	HLA-DQA 1 03:01 /DQB1 03:02	31.56
4	HLA-DRB 1*04:01	15.54	32	HLA-DQA 1 01:02/DQB 1 06:04	19.00
5	HLA-DRB 1*11:01	10.92	33	HLA-DQA 1 05:01 /DQB 1 03:01	80.58
6	HLA-DRB 1*13:01	9.86	34	HLA-DQA1 02:01/DQB 1 02:02	27.99
7	HLA-DRB 1*07:01	19.84	35	HLA-DQA 103:01/DQB 103:01	49.92
8	HLA-DRB 1*01:01	4.06	36	HLA-DQA 1 02:01/DQB1 03:03	23.32
9	HLA-DRB 1*01:02	1.85	37	HLA-DQA 1 03:03/DQB 1 03:03	20.22
10	HLA-DRB 1*04:02	6.28	38	HLA-DP A1 01:03/DPB 101:01	99.83
11	HLA-DRB 1*04:05	1.22	39	HLA-DPA1 01:03/DPB 1 02:01	99.83
12	HLA-DRB 1*04:07	2.78	40	HLA-DP A1 01:03/DPB 103:01	99.82
13	HLA-DRB 1*04:08	1.26	41	HLA-DPA1 01:03/DPB 1 04:01	99.88
14	HLA-DRB 1*08:04	0.86	42	HLA-DPA1 01:03/DPB 1 04:02	99.86
15	HLA-DRB 1*09:01	5.33	43	HLA-DP A1 01:03/DPB 1 05:01	99.81
16	HLA-DRB 1*10:01	2.78	44	HLA-DPAl02:01/DPB 101:01	23.54
17	HLA-DRB 1*11:02	0.94	45	HLA-DPAl02:01/DPB 102:01	24.11
18	HLA-DRB 1*11:03	0.74	46	HLA-DPAl02:01/DPB 103:01	17.63
19	HLA-DRB 1*11:04	4.76	47	HLA-DPAl02:01/DPB 104:01	46.73
20	HLA-DRB 1*15:02	0.78	48	HLA-DPA1 02:01/DPB 1 04:02	38.04
21	HLA-DRB 1*15:03	1.22	49	HLA-DPAl02:01/DPB 105:01	13.24
22	HLA-DRB1*16:O1	4.06	50	HLA-DPAl02:01/DPB 106:01	8.59
23	HLA-DRB1*16:O2	0.84	51	HLA-DPAl02:01/DPB 109:01	7.26
24	HLA-DRB3 *02:02	0.00	52	HLA-DPAl02:01/DPBlll:01	9.98
25	HLA-DRB3*03:01	0.00	53	HLA-DPAl02:01/DPBl13:01	11.55
26	HLA-DRB5*01:01	0.00	54	HLA-DPAl02:01/DPBl14:01	7.98
27	HLA- DQAl01:01/DQB 105:01	30.57	55	HLA-DPAl02:01/DPBl15:01	7.73
28	HLA- DQAl05:01/DQB 102:01	76.17	56	HLA-DPAl02:01/DPBl17:01	10.40

[0285] Algorithm-based T cell epitope prediction tools available from the IEDB were used to predict IC50 values for binding to HLA class I molecules for each 8-14 amino acid peptide in the dataset using ANN (Nielsen et al. (2003) Protein Sci., 12:1007-1017 and Lundegaard et al. (2008) NAR, 36:W509-512) and, in some cases, one or more additional prediction using SMM (Peters and Sette (2005) BMC Bioinformatics, 6:132) and comblib (Sidney et al. (2008) Immunome Res. 4:2, or the Consensus tool (see Kim, et al. (2012) Immune epitope database

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PCT/US2016/064861 analysis resource, NAR (combining predictions from any of the foregoing). Predictions for IC50 values for binding to HLA class II for each 15 amino acid peptide in the dataset was made using the NetMHCIIpan method (Karosiene et al. (2013) Immunogenetics 65(10):711; Nielsen et al. (2008) PLoS Comput Biol.4(7)el000107). Lor each individual position within the portion of the CAR amino acid sequence, the total number of sequences in the dataset that included the position and was predicted to bind to any of the class I or class II alleles with a predicted IC50 of less than 50 nm was determined. FIGs 4A (HLA class I) and 4B (HLA class II), depict the results for class I and class II alleles, respectively, showing positional coverage along the length of the sequence, based on the determined total number, weighted according to the frequency of the individual HLA alleles in the population. The area under the curve (AUC) across the entire assessed region was approximately 1321 for HLA class I binding and 2943 for HLA class II binding. The AUC for the transmembrane-costimulatory domain region was approximately 931 for HLA class I binding and 2212 for HLA class II binding.

[0286] As shown in FIGs. 4A and 4B, certain portions of the sequence were predicted by this method to contain fragments more likely to bind well in MHC complexes and thus be presented as epitopes for potential recognition by T cells. Binding affinity for HLA alleles alone does not necessarily predict immunogenicity. Given that the individual domains (e.g., transmembrane, co stimulatory) in this exemplary CAR were human-derived, upon administration to a human subject, immunogenic responses were less likely to develop against an epitope within any one of these individual regions alone (as opposed to an epitope spanning multiple regions not ordinarily associated with one another, and/or including a junction between such regions). For example, even for a peptide predicted to bind well to and be presented in the context of an MHC molecule, if the peptide was derived entirely from an endogenous protein, it may be recognized as “self’ and thus may fail to induce a productive immune response. For example, whereas certain regions entirely within a single transmembrane or cytoplasmic domain scored highly on the HLA-binding affinity prediction, in the results described in Example 1, no immune responses were detected against peptide sequences solely within either one of these domains of a similar CAR sequence. Accordingly, while various “hot spots” were observed with respect to predicted HLA-binding affinity, subsequent assessment and alteration focused on those areas that not only had higher predicted IC50 values, but also included potential epitopes that spanned the junction between different domains derived from two different proteins.

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PCT/US2016/064861 [0287] In particular, a junction region that includes one or more potential peptide epitopes spanning the junction of the CD28 transmembrane domain and 4-IBB signaling domain of the exemplary CAR was further assessed. With respect to the sequence set forth in SEQ ID NO:5, which includes the exemplary human CD28 transmembrane domain (SEQ ID NO:2) and exemplary human 4-1BB costimulatory domain (SEQ ID NO:3), the assessed junction region contained 13 amino acids on either side of the junction spanning the CD28 transmembrane and 4-IBB costimulatory domains as follows:

FWVLVVVGGVLACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSC RFPEEEEGGCEL (SEQ ID NO:5), in which a 26 amino acid junction region is indicated by bold, and the two amino acids just C’ and N’ of the junction between the domains is indicated by underline. The assessed 26 amino acid junction region is set forth in SEQ ID NO: 137 and corresponds to amino acid residues 15 to 40 of the sequence of amino acids set forth in SEQ ID NO:5.

[0288] In silico modeling was carried out to identify one or more amino acid modifications (mutations) within the 26-amino acid junction region set forth in SEQ ID NO: 137 resulting in peptide fragments that were predicted to bind with high IC50 values to class I and class II alleles, and thus that were likely to reduce the potential for inducing immunogenicity against a CAR containing this region. Specifically, predictions were made for variant peptide fragments of the junction region containing one or more mutations at amino acid residue positions corresponding to positions 14, 17 and 20 with numbering with reference to SEQ ID NO: 137 (which correspond to one or more mutations at amino acid positions corresponding to positions 28, 31 and 34 with numbering with reference to SEQ ID NO:5). In this exemplary study, these residues were chosen for further analysis following in silico mutagenesis and binding predictions of all high affinity epitopes in which all possible single amino acid replacements across that epitope were surveyed for their impact on the predicted IC50 values. Residues that resulted in greater IC50 predictions (decrease in the binding) were identified, which identified the above residues as being sensitive to replacements.

[0289] A series of different variant junction regions were assessed, each containing one or more amino acid replacement at the assessed position(s), as compared to the non-mutated junction region within the exemplary CAR sequence. An exemplary subset of amino acid replacements at the identified positions were chosen that may be less disruptive to the structure or function of either the transmembrane region of the costimulatory signaling domain. Also,

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PCT/US2016/064861 replacements were chosen that may be able to impact more than one epitope at a time, since the epitopes overlap. Specifically, individual variant junction regions contained the following modifications (amino acid replacements): K28A, K28H, K28L, K28Q, K28S, R31A, R31H, R31L, R31N, R31S, L34A, L34S, K28Q/R31A, K28Q/R31N, K28Q/R31S, K28Q/L34A, K28Q/L34S, R31N/L34A, R31N/L34S, K28Q/R31N/L34A, K28Q/R31N/L34S, with numbering with reference to SEQ ID NO:5.

[0290] For the non-variant and variant junction regions, weighted immunogenicity scores were obtained for class I and class II alleles, using the T cell epitope prediction tools available from IEDB. Scores were derived using predicted IC50 values for each of a series of 8-mer to

14- mer overlapping peptides (for each of the 27 HLA class I alleles, individually) and a series of

15- mer overlapping peptides (for each of the 56 HLA class II alleles, individually) within the respective (variant or non-variant) 26-amino acid junction region, and were weighted based on relative frequency in the population of the individual HLA class I and class II alleles. A higher relative score is indicative of a higher degree of predicted binding.

[0291] The results are set forth in FIG. 5. The results demonstrate the ability to decrease in the overall predicted HLA class I immunogenicity score within a CAR junction region by modifying amino acids within the region. The results also confirm the ability to reduce predicted HLA class I binding affinity (and hence reduced predicted immunogenicity score) without resulting in a substantial increase in the predicted immunogenicity score for HLA class II binding. Thus, in general, the results showed that amino acid modification(s) within a region spanning a junction between a CD28 transmembrane domain and a 4-IBB costimulatory domain of a CAR could be made and effect an overall reduction in the predicted affinity for human HLA binding, which would be consistent with a reduction in potential for immunogenicity, upon administration to a human subject, of a chimeric receptor identical to a receptor having this region, but containing the modification or combination of modifications in this region.

Example 3: Comparison of In Silico Analysis and In Vitro Binding of Peptides Derived from Junction Regions of a CAR for Binding to HLA Class I [0292] Actual binding affinities for certain HLA class I alleles (A*02:01, A*03:01,

A*ll:01, and B*08:01) were assessed in vitro for exemplary overlapping 9 amino acid peptide sequences within a portion of the 26 amino acid junction region spanning the CD28 and 4-IBB junction. Specifically, assessment was of a series of overlapping 9-mer peptides derived from

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PCT/US2016/064861 the sequence VAFIIFWVKRGRKKLL (set forth in SEQ ID NO: 7), which contains a portion of the CD28 transmembrane domain and 4-IBB costimulatory domain spanning the junction between the domains (bond joining the two amino acids noted in underline). In addition, a series of overlapping 9-mer peptides of each of a number of different variants of this portion also were assessed, each variant containing a mutation or mutations in this region as described in Example 2.

[0293] The various 9-mer overlapping peptides were synthesized and their purity tested by MALDI-TOF Mass Spectrometry. The synthetic peptides were then incubated with recombinant MHC molecules to assess binding properties using the REVEAL Epitope Discovery System, which is a high-throughput binding assay that measures the degree to which each peptide is able to stabilize a ternary MHC-peptide complex (ProImmune, Oxford, United Kingdom). Each peptide was separately tested for this ability with respect to each of the HLA class I alleles, normalized to the degree observed for a positive control (known T cell epitope for the relevant allele). The results are reported as a score, in which the binding was normalized to the positive control peptide set at 100%. In this analysis, a score of greater than 50 generally was considered to represent good or high affinity binding.

[0294] The results were compared to predicted binding (IC50) values obtained for binding of the same peptide:MHC complex, using the in silico prediction methods as described in Example 2. Since the maximum IC50 value predicted was about 50,000, the IC50 values were log transformed, subtracted from LOG(50000) and divided by LOG(50000) to obtain a normalized in silico score ((Log(50000) - logIC50) / Log(50000)). In this analysis, an in silico binding prediction score of greater than 2.0 generally was considered to represent predicted good or high affinity binding.

[0295] The results are set forth in Table 2A (HLA-A*02:01 and HLA-A*03:01) and Table 2B (HLA-A*ll:01 and HLA-B*08:01). In general, the in silico binding predictions were predictive of the actual in vitro binding results. In some cases, a relatively higher binding was predicted in silico, but not observed in the in vitro assay.

[0296] The results also were consistent with predicted binding affinity being generally predictive of affinity as measured in the in vitro assay. Additionally, the results demonstrated successful reduction of binding affinity to an HLA by modifications within a junction region, and that it was possible to modify the sequence in a way that resulted in a lower predicted or actual binding affinity or score of one of the overlapping potential epitopes, without increasing

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PCT/US2016/064861 (or while also reducing) binding affinity or score for another of the overlapping epitopes containing the same residue. In some embodiments, such mutations or modifications may be particularly advantageous. As a non-limiting example of the results, modifications K28L, R31H, L34S and/or L34A, with reference to numbering set forth in SEQ ID NO:5, generally resulted in a reduced predicted or actual binding affinity or score for at least one HLA allele and/or for at least one peptide within the region assessed, without resulting in a higher binding affinity to another HLA allele and/or without resulting in a higher binding affinity for another peptide.

Table 2A: In Silico and In \	itro MHC binding of Variant Peptides
			A*02:01	A*03:01
Over- lapping Reference Peptide	Peptide Sequence	Mutation	IEDB IC50	In Silico Score	In Vitro Score	IEDB IC50	In Silico	In Vitro	SEQ ID NO.
1	VAFIIFWVK	none	19617	0.41	0.70	262	2.28	1.50	16
2	AFIIFWVKR	none	25314	0.30	0.50	17846	0.45	5.70	17
3	FIIFWVKRG	none	7967	0.80	10.10	23693	0.32	1.40	18
4	IIFWVKRGR	none	24769	0.31	1.70	406	2.09	24.30	19
5	IFWVKRGRK	none	30463	0.22	4.50	3482	1.16	20.50	20
6	FWVKRGRKK	none	28878	0.24	3.50	17327	0.46	2.20	21
7	WVKRGRKKL	none	27956	0.25	1.40	22961	0.34	13.10	22
1	VAFIIFWVS	K28S	12273	0.61	70.60	22660	0.34	0.70	23
2	AFIIFWVSR	K28S	23924	0.32	4.50	18157	0.44	0.30	24
3	FIIFWVSRG	K28S	3382	1.17	0.20	21751	0.36	0.10	25
4	IIFWVSRGR	K28S	21442	0.37	2.60	155	2.51	25.30	26
5	IFWVSRGRK	K28S	30615	0.21	1.30	1880	1.42	39.20	27
6	FWVSRGRKK	K28S	28679	0.24	1.50	17832	0.45	2.90	28
7	WVSRGRKKL	K28S	23551	0.33	2.30	22394	0.35	2.10	29
1	VAFIIFWVL	K28L	1336	1.57	0.20	20145	0.39	0.00	30
2	AFIIFWVLR	K28L	22444	0.35	5.70	15583	0.51	0.30	31
3	FIIFWVLRG	K28L	2037	1.39	7.20	20853	0.38	0.10	32
4	IIFWVLRGR	K28L	17613	0.45	8.40	238	2.32	2.80	33
5	IFWVLRGRK	K28L	30293	0.22	2.90	3675	1.13	19.50	34
6	FWVLRGRKK	K28L	28857	0.24	3.30	16996	0.47	0.40	35
7	WVLRGRKKL	K28L	19522	0.41	2.40	23063	0.34	0.70	36
1	VAFIIFWVH	K28H	23252	0.33	3.10	10359	0.68	0.30	37
2	AFIIFWVHR	K28H	22819	0.34	0.30	18506	0.43	0.30	38
3	FIIFWVHRG	K28H	1691	1.47	37.30	22930	0.34	0.00	39
4	IIFWVHRGR	K28H	23573	0.33	4.70	326	2.19	30.10	40
5	IFWVHRGRK	K28H	29930	0.22	7.10	1062	1.67	37.80	41
6	FWVHRGRKK	K28H	28189	0.25	1.60	17052	0.47	3.60	42
7	WVHRGRKKL	K28H	25035	0.30	1.60	22278	0.35	3.40	43
1	VAFIIFWVA	K28A	2733	1.26	28.50	21010	0.38	0.00	44

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2	AFIIFWVAR	K28A	23072	0.34	2.20	17755	0.45	0.50	45
3	FIIFWVARG	K28A	1902	1.42		22486	0.35		102
4	IIFWVARGR	K28A	22077	0.36	9.60	206	2.39	18.60	46
5	IFWVARGRK	K28A	30251	0.22	1.00	3893	1.11	53.70	47
6	FWVARGRKK	K28A	28353	0.25	13.50	16210	0.49	5.00	48
7	WVARGRKKL	K28A	22630	0.34	8.80	22548	0.35	1.30	49
1	VAFIIFWVQ	K28Q	17921	0.45	5.30	22927	0.34	0.20	50
2	AFIIFWVQR	K28Q	24165	0.32	0.40	19279	0.41	0.20	51
3	FIIFWVQRG	K28Q	4152	1.08	16.80	23136	0.33	0.40	52
4	IIFWVQRGR	K28Q	21783	0.36	5.80	231	2.34	18.00	53
5	IFWVQRGRK	K28Q	30704	0.21	6.10	3177	1.20	36.70	54
6	FWVQRGRKK	K28Q	29501	0.23	5.20	18619	0.43	6.40	55
7	WVQRGRKKL	K28Q	24480	0.31	8.10	23630	0.33	4.90	56
4	IIFWVKRGS	R31S	18902	0.42	13.60	19450	0.41	16.20	57
5	IFWVKRGSK	R31S	29391	0.23	2.40	3348	1.17	10.40	58
6	FWVKRGSKK	R31S	28317	0.25	1.90	14227	0.55	5.90	59
7	WVKRGSKKL	R31S	23485	0.33	3.00	22637	0.34	1.90	60
4	IIFWVKRGL	R31L	2692	1.27	82.20	16661	0.48	15.90	61
5	IFWVKRGLK	R31L	29250	0.23	2.40	1973	1.40	26.90	62
6	FWVKRGLKK	R31L	27554	0.26	9.40	14434	0.54	2.70	63
7	WVKRGLKKL	R31L	20709	0.38	24.50	22985	0.34	10.00	64
4	IIFWVKRGH	R31H	27453	0.26	5.30	1665	1.48	26.60	65
5	IFWVKRGHK	R31H	29806	0.22	2.20	3806	1.12	9.00	66
6	FWVKRGHKK	R31H	27689	0.26	2.60	16743	0.48	4.10	67
7	WVKRGHKKL	R31H	25923	0.29	3.90	22313	0.35	1.40	68
4	IIFWVKRGA	R31A	6107	0.91	85.90	16069	0.49	42.90	69
5	IFWVKRGAK	R31A	29354	0.23	13.80	3470	1.16	22.00	70
6	FWVKRGAKK	R31A	28151	0.25	4.30	17066	0.47	4.70	71
7	WVKRGAKKL	R31A	24746	0.31	4.70	22982	0.34	4.10	72
4	IIFWVKRGN	R31N	25979	0.28	9.30	18552	0.43	4.30	73
5	IFWVKRGNK	R31N	29978	0.22	3.50	2669	1.27	8.00	74
6	FWVKRGNKK	R31N	28430	0.25	8.70	17713	0.45	3.60	75
7	WVKRGNKKL	R31N	24790	0.30	2.40	22813	0.34	0.90	76
4	IIFWVQRGS	K28Q/R3 IS	14326	0.54	22.40	17741	0.45	4.50	77
5	IFWVQRGSK	K28Q/R3 IS	29540	0.23	33.50	3052	1.21	20.30	78
6	FWVQRGSKK	K28Q/R3 IS	28907	0.24	0.80	15992	0.50	1.90	79
7	WVQRGSKKL	K28Q/R3 IS	18121	0.44	4.40	23279	0.33	1.20	80
4	IIFWVQRGA	K28Q/R3 1A	2658	1.27	95.30	13467	0.57	2.70	81
5	IFWVQRGAK	K28Q/R3 1A	29482	0.23	11.70	3205	1.19	17.00	82
6	FWVQRGAKK	K28Q/R3 1A	28792	0.24	2.20	18649	0.43	2.70	83

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7	WVQRGAKKL	K28Q/R3 1A	20217	0.39	3.00	23651	0.33	2.00	84
4	IIFWVQRGN	K28Q/R3 IN	23103	0.34	13.00	16590	0.48	3.60	85
5	IFWVQRGNK	K28Q/R3 IN	30164	0.22	16.10	2438	1.31	23.20	86
6	FWVQRGNKK	K28Q/R3 IN	29113	0.23	3.10	19165	0.42	1.30	87
7	WVQRGNKKL	K28Q/R3 IN	19790	0.40	4.30	23457	0.33	1.50	88
7	WVKRGRKKS	L34S	30812	0.21	3.90	25365	0.29	0.90	89
7	WVKRGRKKA	L34A	28556	0.24	4.50	24086	0.32	0.90	90
7	WVQRGNKKS	K28Q/L3 4S	26883	0.27	1.20	25680	0.29	0.70	91
7	WVQRGNKKA	K28Q/L3 4A	21998	0.36	1.90	24564	0.31	1.20	92
1	VAFIIFWVR	K28R	20045	0.40	1.20	5746	0.94	0.70	100
2	AFIIFWVRR	K28R	25059	0.30	1.40	17924	1.00		101

Table 2B: In Silico and In Vitro MHC binding of Variant Peptides
			A*ll:01	B*08:01
Overlapping Reference Peptide	Peptide Sequence	Mutation	IEDB IC50	In Silico Score	In Vitro Score	IEDB IC50	In Silico Score	In Vitro Score	SEQ ID NO.
1	VAFIIFWVK	wt	19	3.42	109.20	15806	0.50	0.30	16
2	AFIIFWVKR	wt	3271	1.18	25.90	23244	0.33	1.90	17
3	FIIFWVKRG	wt	22946	0.34	0.40	18081	0.44	0.10	18
4	IIFWVKRGR	wt	706	1.85	54.50	23025	0.34	0.00	19
5	IFWVKRGRK	wt	9701	0.71	29.30	23513	0.33	0.40	20
6	FWVKRGRKK	wt	22175	0.35	6.50	21764	0.36	0.00	21
7	WVKRGRKKL	wt	24020	0.32	4.80	225	2.35	28.50	22
1	VAFIIFWVS	K28S	12403	0.61	25.10	13796	0.56	0.10	23
2	AFIIFWVSR	K28S	2175	1.36	54.00	21862	0.36	0.30	24
3	FIIFWVSRG	K28S	20651	0.38	5.10	17854	0.45	0.40	25
4	IIFWVSRGR	K28S	162	2.49	68.90	23445	0.33	0.00	26
5	IFWVSRGRK	K28S	5778	0.94	39.20	23448	0.33	0.00	27
6	FWVSRGRKK	K28S	21126	0.37	6.30	23067	0.34	0.00	28
7	WVSRGRKKL	K28S	23684	0.32	4.40	3208	1.19	6.20	29
1	VAFIIFWVL	K28L	14191	0.55	0.20	1196	1.62	0.00	30
2	AFIIFWVLR	K28L	850	1.77	17.20	23012	0.34	0.20	31
3	FIIFWVLRG	K28L	19162	0.42	2.40	17427	0.46	0.20	32
4	IIFWVLRGR	K28L	185	2.43	35.20	23298	0.33	0.40	33
5	IFWVLRGRK	K28L	6386	0.89	29.30	23324	0.33	0.00	34
6	FWVLRGRKK	K28L	21862	0.36	5.00	23437	0.33	0.00	35
7	WVLRGRKKL	K28L	23725	0.32	3.20	1342	1.57	4.00	36

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1	VAFIIFWVH	K28H	1209	1.62	12.30	16202	0.49	0.00	37
2	AFIIFWVHR	K28H	4939	1.01	34.90	22682	0.34	0.30	38
3	FIIFWVHRG	K28H	19850	0.40	0.90	17464	0.46	0.20	39
4	IIFWVHRGR	K28H	196	2.41	77.50	22662	0.34	0.20	40
5	IFWVHRGRK	K28H	7133	0.85	36.60	22030	0.36	0.10	41
6	FWVHRGRKK	K28H	22214	0.35	5.60	23778	0.32	0.00	42
7	WVHRGRKKL	K28H	23844	0.32	5.60	814	1.79	19.20	43
1	VAFIIFWVA	K28A	12499	0.60	2.80	5131	0.99	0.00	44
2	AFIIFWVAR	K28A	2784	1.25	51.80	21850	0.36	0.20	45
3	FIIFWVARG	K28A	20922	0.38		18463	0.43		102
4	IIFWVARGR	K28A	239	2.32	70.60	23580	0.33	0.30	46
5	IFWVARGRK	K28A	8772	0.76	34.90	23612	0.33	0.00	47
6	FWVARGRKK	K28A	20762	0.38	8.90	23035	0.34	0.00	48
7	WVARGRKKL	K28A	23920	0.32	13.80	2821	1.25	32.40	49
1	VAFIIFWVQ	K28Q	15477	0.51	3.90	14875	0.53	0.20	50
2	AFIIFWVQR	K28Q	2174	1.36	15.00	23016	0.34	0.00	51
3	FIIFWVQRG	K28Q	22161	0.35	1.50	17653	0.45	0.60	52
4	IIFWVQRGR	K28Q	361	2.14	72.80	23548	0.33	0.10	53
5	IFWVQRGRK	K28Q	9561	0.72	128.50	23670	0.32	0.00	54
6	FWVQRGRKK	K28Q	22394	0.35	5.70	22604	0.34	1.90	55
7	WVQRGRKKL	K28Q	23688	0.32	12.80	2512	1.30	45.10	56
4	IIFWVKRGS	R31S	18923	0.42	100.00	22325	0.35	0.40	57
5	IFWVKRGSK	R31S	7476	0.83	47.90	21691	0.36	1.20	58
6	FWVKRGSKK	R31S	19910	0.40	5.60	20823	0.38	0.10	59
7	WVKRGSKKL	R31S	24090	0.32	11.80	585	1.93	56.10	60
4	IIFWVKRGL	R31L	20182	0.39	32.40	17368	0.46	1.70	61
5	IFWVKRGLK	R31L	4201	1.08	61.10	23537	0.33	58.30	62
6	FWVKRGLKK	R31L	17309	0.46	25.30	20839	0.38	6.30	63
7	WVKRGLKKL	R31L	24095	0.32	22.10	765	1.82	65.70	64
4	IIFWVKRGH	R31H	7117	0.85	19.50	23227	0.33	0.10	65
5	IFWVKRGHK	R31H	10783	0.67	30.90	23461	0.33	3.20	66
6	FWVKRGHKK	R31H	17635	0.45	27.60	20754	0.38	0.40	67
7	WVKRGHKKL	R31H	23924	0.32	2.80	269	2.27	47.60	68
4	IIFWVKRGA	R31A	19134	0.42	22.60	19585	0.41	0.90	69
5	IFWVKRGAK	R31A	8311	0.78	69.70	21592	0.36	2.20	70
6	FWVKRGAKK	R31A	20234	0.39	13.20	21762	0.36	0.30	71
7	WVKRGAKKL	R31A	23857	0.32	4.60	1366	1.56	34.10	72
4	IIFWVKRGN	R31N	19351	0.41	42.10	22864	0.34	0.90	73
5	IFWVKRGNK	R31N	6780	0.87	45.90	24238	0.31	0.10	74
6	FWVKRGNKK	R31N	20732	0.38	7.30	21565	0.37	0.10	75
7	WVKRGNKKL	R31N	24036	0.32	4.10	1181	1.63	65.90	76
4	IIFWVQRGS	K28Q/R31S	18031	0.44	16.90	22961	0.34	0.10	77
5	IFWVQRGSK	K28Q/R31S	7300	0.84	103.30	22846	0.34	0.00	78
6	FWVQRGSKK	K28Q/R31S	20419	0.39	6.80	21853	0.36	0.00	79
7	WVQRGSKKL	K28Q/R31S	23740	0.32	5.90	5230	0.98	6.80	80

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4	IIFWVQRGA	K28Q/R31A	18055	0.44	56.50	20759	0.38	0.20	81
5	IFWVQRGAK	K28Q/R31A	8237	0.78	61.30	22801	0.34	0.20	82
6	FWVQRGAKK	K28Q/R31A	20696	0.38	18.30	22612	0.34	0.00	83
7	WVQRGAKKL	K28Q/R31A	23552	0.33	3.80	8314	0.78	11.40	84
4	IIFWVQRGN	K28Q/R31N	18437	0.43	29.00	23425	0.33	0.00	85
5	IFWVQRGNK	K28Q/R31N	6566	0.88	67.50	24059	0.32	0.10	86
6	FWVQRGNKK	K28Q/R31N	21228	0.37	5.90	22436	0.35	0.00	87
7	WVQRGNKKL	K28Q/R31N	23751	0.32	10.70	7869	0.80	32.00	88
7	WVKRGRKKS	L34S	23906	0.32	6.70	10345	0.68	4.30	89
7	WVKRGRKKA	L34A	23864	0.32	3.10	1225	1.61	3.10	90
7	WVQRGNKKS	K28Q/L34S	23612	0.33	5.00	21235	0.37	0.00	91
7	WVQRGNKKA	K28Q/L34A	23576	0.33	0.80	14247	0.55	0.70	92
1	VAFIIFWVR	K28R	108	2.67	34.60	16290	0.49	0.10	100
2	AFIIFWVRR	K28R	3387	1.17	1.50	22717	0.34	0.30	101

Example 4: Analysis of Peptides Derived from Junction Region of a CAR for Binding to HLA-A2:01 [0297] In order to identify CAR-derived peptides potentially capable of inducing immunogenic responses, a series of overlapping peptides within the non-variant (reference) sequence containing the junction between the CD28 transmembrane domain and 4-IBB costimulatory domain of a CAR were assessed in silico. Algorithms were used to predict binding affinities for the peptide groove of a common human MHC class I molecule (HLA-A2:01) using in silico analysis to predict affinity for binding. As set forth in FIG. 3, the assessed portion of the CAR had the sequence CYSLLVTVAFIIFWVKRGRKKLLYIFKQPF (set forth in SEQ ID NO: 6), which contains a portion of the of the CD28 transmembrane domain (set forth in SEQ ID NO:2) and a portion of the 4-IBB costimulatory domain (set forth in SEQ ID NO:3), with the residues spanning the junction of the domains shown by underline. Predicted HLA-A2:01 binding affinity was assessed in silico for a series of 140 overlapping peptides of 8-14 amino acids of the sequence set forth in SEQ ID NO:6. Thirty-five (35) of the peptides contained only sequence from the transmembrane domain portion; 35 of the peptides contained only from the costimulatory domain portion, and 70 of the peptides had a junction or fusion region sequence, containing amino acid residues bridging the junction between the domains. For this assessment, peptide fragments predicted to bind to HLA-A2:01 with a dissociation constant of 0 nM to 50 nM were considered predicted to bind with high affinity. Peptide fragments predicted to bind with a dissociation constant of 51 nM to 1000 nM were considered predicted to bind with low

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[0298] As shown in FIG. 3, two of the peptides derived from the reference sequence in this region, each containing a sequence with an overlapping region spanning the junction between the domains were predicted to exhibit low binding affinity for HLA-A2:01. Specifically, a 14mer peptide having the sequence FIIFWVKRGRKKLL (SEQ ID NO: 10), was predicted to bind with a dissociation constant of 294 nM, and a 13-mer peptide having the sequence of FIIFWVKRGRKKL (SEQ ID NO: 11) was predicted to bind with a dissociation constant of 618 nM. These peptides each included a portion of the 15-mer peptide set forth in SEQ ID NO:8 and identified in Example 1. Shorter 8-mer to 12-mer peptides within this sequence were not predicted to exhibit binding to HLA-A2:01. Another 13-mer peptide containing the amino acid sequence IIFWVKRGRKKLL (SEQ ID NO: 12) was predicted to have a rare binding affinity with a predicted dissociation constant of approximately 3000 nM. None of the remaining fragments that bridged the junction between the two domains were predicted by this assay to exhibit binding affinity for HLA-A2:01 (all had a predicted dissociation constant of far greater than 5000 nM, and in most cases higher than 14,000 nm or 20,000 nM or greater). In each of the peptides predicted to bind to HLA-A2:01, neither of the two junction-spanning residues (VK) themselves was predicted to be an anchor residue; rather, such peptides contained these residues in non-flanking positions.

[0299] Approximately 15 of the peptides containing sequence derived only from the transmembrane domain were predicted to have a dissociation constant for HLA-A2:01 of less than 5000 nM. Two peptides containing sequence only from the co-stimulatory domain were predicted to have a dissociation constant for HLA-A2:01 binding of less than 5000 nM. The costimulatory domain and transmembrane domain in the assessed sequence are derived from endogenous human sequences, which generally are less likely to be immunogenic to a human subject. For example, in the study described in Example 1, no immune responses were detected that were specific for peptide sequences solely within either one of these domains of the CAR. Accordingly, variants of peptides containing sequence spanning the junction region were assessed.

[0300] To generate variant peptides predicted to have reduced binding affinities to HLAA2:01 and/or reduced immunogenicity in a human subject having this HLA allele, a variant sequence was generated in silica, containing mutations in the junction region as compared to the

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PCT/US2016/064861 sequence set forth in SEQ ID NO:6. Given that peptides containing the junction-spanning “VK” residues (at non-anchor positions) were predicted to exhibit high binding affinities for HLAA2:01, two asparagine residues were inserted in the junction between the CD28 transmembrane and 4-IBB co-stimulatory domains. The variant contained the sequence

CYSLLVTVAFIIFWVNNKRGRKKLLYIFKQPF (set forth in SEQ ID NO: 13, the sequence flanking the junction that was generated by insertion of the asparagine residues is shown in underline). The exemplary variant sequence of SEQ ID NO: 13 was assessed by the same predictive methods. To assess predicted binding affinities for this variant sequence, a series of 154 overlapping fragments of 8-14 amino acids of the sequence set forth in SEQ ID NO: 13 were assessed by in silico analysis as described above, whereby 35 peptides had a sequence only in the transmembrane portion, 35 peptides had a sequence only in the costimulatory domain portion and 84 peptides contained a junction region sequence containing amino acids bridging the domains, including one or both of the inserted asparagine residues.

[0301] The results are depicted in FIG. 3. As shown, overall, the HLA-A2:01 binding affinities of overlapping peptides within the variant region containing the junction, collectively, were substantially reduced as compared to the non-variant sequence. In particular, the predicted dissociation constant for binding to HLA-A2:01 of peptides in the portion of the junction region previously predicted to be immunogenic was substantially reduced. For example, peptide variants IIFWVNNKRGRKKL(SEQ ID NO: 14) and IIFWVNNKRGRKK (SEQ ID NO: 15), which included asparagine residues in the altered region flanking the junction compared to peptides identified as set forth in SEQ ID NOS: 10 and 11, respectively, were predicted to exhibit no detectable binding affinity to HLA-A2:01. Two 14-mer peptides, FIIFWVNNKRGRKK (SEQ ID NO:96) and IFWVNNKRGRKKLL (SEQ ID NO:97), were predicted to exhibit a dissociation constant for binding to this HLA indicating a rare binding affinity, within the range of 1000 nM to 5000 nM. All other peptides containing the modified junction region sequence were predicted to exhibit a dissociation constant of greater than 5000 nM, and in most cases higher than 14,000 nM or 20,000 nM or greater, and thus were not predicted to exhibit binding affinity for HLA-A2:01 by this assessment. Additionally, the modification of the junction region sequence did not create any new peptides predicted to have higher binding affinities for HLA-A2:01 within the costimulatory or transmembrane domain regions.

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Example 5: Administration of anti-CD22 CAR-Expressing Cells to Subjects Previously Treated with anti-CD19 CAR [0302] Six subjects with relapsed/refractory CD22⁺ B cell acute lymphoblastic leukemia (ALL) were administered autologous T cells expressing an anti-CD22 chimeric antigen receptor (CAR). The CAR included a human anti- CD22 scFv antibody, a CD8alpha transmembrane domain, a 4-IBB intracellular signaling domain, and a CD3zeta intracellular signaling domain.

[0303] All subjects had previously undergone at least one prior allogeneic hematopoietic stem cell transplant and had received treatment with one of various CD19-directed CAR-T cell therapies. Five of the subjects had relapsed with ALL on which CD 19 was not detected (“CD 19 neg”) and one subject was otherwise a non-responder to the prior CD 19 CAR therapy.

[0304] Table 3 summarizes the characteristics of the treated patients.

Table 3: Patient Characteristics

ID	Age/Sex	Prior HCT	Prior antiCD19 CAR	CD19 neg relapse	CD22 site density	Pre-HCT disease burden (% leukemia in aspirate)
1	22/M	Y	Y	Y	2084	>95%
2	20/F	Y(2)	Y	Y	13452	5%
3	22/M	Y	Y	Y	846	>90%
4	22/M	Y	Y	N	2589	95%
5	7/F	Y	Y	Y	2839	32%
6	17/F	Y	Y	Y	2185	1%

HCT: hematopoietic cell transplantation.

[0305] Prior to administration of the cells, patients underwent autologous leukapheresis to harvest peripheral blood mononuclear cells (PBMCs). T cells were isolated from the harvested PBMCs by immunoaffinity-based enrichment for CD3 expression and cultured in the presence of anti-CD3/-CD28 beads, followed by transduction with a lentiviral vector encoding the antiCD22 CAR. The cells were cultured for 7-10 days. Subjects received induction chemotherapy

2 with 25 mg/m fludarabine on Days -4, -3 and -2 and 900 mg/m cyclophosphamide on day -2 (cell infusion on Day 0). Each patient received an initial CAR T cell dose of 3 x 10 transduced T-cells/recipient weight (kg) by intravenous infusion. The second subject enrolled developed grade 3 diarrhea, meeting the criteria for dose-limiting toxicity (DLT), which led to dose expansion at the first dose-level to treat a total of 6 subjects. No subsequent DLTs were seen at this dosage. Two subjects developed grade 1 cytokine release syndrome (CRS), one subject developed grade 2 CRS, and in two subjects CRS was not present.

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PCT/US2016/064861 [0306] The number of CAR-T cells in peripheral blood, bone marrow or cerebrospinal fluid was determined at certain timepoints post-treatment by incubating cells with CD22-Fc. For patients in which expansion was observed, evidence for CAR-T cell expansion was seen in peripheral blood, bone marrow and cerebrospinal fluid, beginning at about day 7. The maximum or peak CAR-T cell expansion was generally observed between about day 12 and about day 15 post-infusion. Table 7 sets forth the maximum or peak percentage of anti-CD22 CAR-T cells observed in this assessment period as a percentage of total T cells in each sample for the treated subjects. Clinical responses were evaluated at day 28 (+/- 4 days) post-infusion.

[0307] As shown in Table 4, the results were consistent with responses being generally correlated to degree of CAR-T cell expansion. For three subjects that exhibited no or low CART cell expansion also showed evidence of disease progression. Two other subjects had stable disease, and one was observed with complete remission with no MRD. Flow cytometric CAR persistence was detected out to 47 days post-infusion in this subject, with remission maintained for 3 months post-infusion. The results demonstrate safe, feasible, and clinically active antiCD22 CAR T-cell therapy in subjects having undergone (and having become non-responsive to, e.g., due to epitope/antigen loss) previous anti-CD19 CAR therapy.

Table 4: Treatment response
ID	Maximum CAR expansion (flow)	CRS	Best Response
PB	Marrow	CSF
1	0	0	n/a	None	PD
2	52.3%	19.5%	0%	Gr 1	MRD neg CR
3	73%	36%	32%	Gr 1	SD
4	6%	1%	0%	Gr2	SD
5	0%	1.3%	0%	None	PD
6	1.8%	2%	0%	None	PD

PB: peripheral blood; CSF: cerebrospinal fluid; CRS: cytokine release syndrome; PD: progressive disease: MRD: minimal residual disease; CR: complete remission; SD: stable disease.

Example 6: Comparison of 7n Siiico Analysis and 7// Vitro Binding of Peptides Derived from Junction Regions of a CAR for Binding to HLA Class II [0308] Actual binding affinities for certain HLA class II alleles (DPAl*01:03;DPBl*04:01, DRA*01:01;DRB 1*03:01, DRA*01:01, DRBl*15:01, DRA*01:01;DRBl*ll:01, DPAl*01:03;DPBl*04:02, DPA 1 *01:03 ;DPB 1*03:01, DPA 1 *01:03;DPB 1*01:01,

DPA 1 *02:01;DPB 1*01:01, DPAl*02:01;DPBl*04:02, DRA*01:01, DRB1*11:O4, DRA*01:01, DRBl*01:02, and DPAl*02:01;DPBl*15:01) were assessed in vitro for

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PCT/US2016/064861 exemplary overlapping 15 amino acid peptide sequences within a portion of the region spanning the junction between the CD28 and 4-lBB-derived sequences of the CAR described in Example 4. Specifically, assessment was of a series of overlapping 15-mer peptides derived from the sequence CYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQT (set forth in SEQ ID NO: 160), which contains a portion of the CD28-derived transmembrane domain and 4-lBB-derived costimulatory domain and spans the junction between the domains (bond joining the two amino acids noted in underline). In addition, a series of overlapping 15-mer peptides of each of a number of different variants of this portion also were assessed, each variant containing a mutation or mutations in this region as described in Example 2.

[0309] The various 15-mer overlapping peptides were synthesized and their purity tested by MAFDI-TOF Mass Spectrometry. The synthetic peptides were then incubated with recombinant MHC molecules to assess binding properties using the REVEAF Epitope Discovery System as described in Example 3. Each peptide was separately tested for this ability with respect to each of the HFA class II alleles, normalized to the degree observed for a positive control (known T cell epitope for the relevant allele). A score was calculated for each allele, in which the binding was normalized to the positive control peptide set at 100%. The potential impact in the population based on these scores was calculated as the sum of the individual allele scores for each peptide.

[0310] The results are set forth in FIG. 7. As shown, the results demonstrated that modifications within the junction region resulted in successful reduction for HFA class II binding of at least one peptide within the region.

[0311] For overlapping 15-mer peptides present within the native junction region, the results were compared to predicted binding (IC50) values obtained for binding of the same peptide:MHC complex, using the in silico prediction methods as described in Example 2. Since the maximum IC50 value predicted was about 50,000, the IC50 values were log transformed, subtracted from FOG(50000) and divided by FOG(50000) to obtain a normalized in silico score ((Fog(50000) - logIC50) / Fog(50000)). Immunogenicity scores (weighted based on relative frequency in the population of the individual HFA class II alleles) were obtained for the normalized in silico and actual binding affinities. The results are set forth in FIG. 8. In general, the in silico binding predictions were predictive of, and in some cases were over-predictive of, the actual in vitro binding results.

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Example 7: Engineering and Characterization of T Cells Transduced with CARs Having Modified Junction Regions [0312] CAR-engineered T cells were generated by transduction of primary T cells with a lentiviral vector encoding either (1) the original chimeric antigen receptor (CAR) as described in Example 1, containing an anti-CD19 scFv, a hinge domain, the junction region set forth in SEQ ID NO :5 (including a transmembrane domain derived from the native CD28 and an intracellular signaling domain derived from the native 4-IBB) (deemed the “native” junction region) and a CD3-zeta intracellular signaling domain or (2) one of a number of particular variants thereof, individually containing a modified junction region in which one or more mutations were introduced in the junction region spanning the junction between the CD28 transmembrane domain and 4-IBB costimulatory signaling domain. Each of the particular variant CARs contained a modified junction region having a mutation (with reference to SEQ ID NO:5) selected from among: K28Q/R31N/L34S, K28Q/R31N/L34A, R31N/L34S, R31N/L34A, K28Q/L34A, K28Q/R31S, K28Q/R31N, K28Q/R31A, L34S, L34A, R31S, R31N, R31A, K28S, K28Q, K28L, K28H, and K28A.

[0313] Primary human CD4+ and CD8+ T cells were isolated by immunoaffinity-based selection from human PBMC samples obtained from healthy donors. The resulting cells were stimulated by culturing with an anti-CD3/anti-CD28 reagent prior to engineering with the respective CAR. Cells were transduced using a lentiviral vector containing a nucleic acid molecule encoding the CAR and a nucleic acid encoding a truncated EGFR (EGFRt), for use as a surrogate marker for transduction, separated by a sequence encoding a T2A ribosome switch.

A mock transduction was used as negative control. Transduced cells were used for expression analysis and cytolytic activity assay.

A. CAR Expression [0314] Cell surface CAR expression (as indicated via the surrogate marker) was assessed at day 17 post-transduction with nucleic acid encoding the native CAR or each of the modified CARs. Cells were stained with anti-EGFR antibody to detect EGFRt as a surrogate to verify CAR expression and assessed by flow cytometry. CAR expression was similar in cells transduced to express the various variant CARs containing a modified junction region, and was comparable to expression of the CAR containing the native junction region.

B. Cytolytic Activity

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PCT/US2016/064861 [0315] Cells engineered to express the native or variant CARs were assessed for cytolytic activity against K562 target cells expressing the CD 19 antigen (K562-CD19). The T cells were incubated with the target cells (K562-CD19) at various effector:target ratios (CAR:Target ratios of 1:1, 2:1 and 4:1) in a well of a culture plate. Lytic activity of the engineered T cells was assessed by measuring the number of caspase-positive target cells per well. The results showed that in this study, the T cells expressing the various variant CARs containing the modified junction region were able to kill CD19-expressing target cells in a target-specific manner, to a similar degree as T cells expressing the CAR containing the “native” junction region.

Example 8: Assessment of Surface Expression and Functionality of CARs Having Modified Junction Regions [0316] CAR-engineered T cells were generated by transduction of primary T cells with a nucleic acid encoding either (1) the original chimeric antigen receptor (CAR) described in Example 1 containing an anti-CD 19 scFv, a hinge domain, the native junction region set forth in SEQ ID NO:5 (including a transmembrane domain derived from the native CD28 and an intracellular signaling domain derived from the native 4-IBB), or (2) a variant thereof containing a modified junction region having a mutation selected from among: K28L, R31H, L34A, or L34S with reference to SEQ ID NO:5. Cell surface CAR expression and functional properties of T cells engineered with CARs containing a modified junction region were assessed.

A. Surface Expression [0317] To directly assess surface expression of the CARs, CAR-engineered T cells were stained with an anti-idiotype antibody specific for the anti-CD 19 scFv contained in the CAR.

The engineered cells also were assessed for surface expression of the surrogate marker EGFRt using an anti-EGFR antibody. To detect intracellular expression of the CARs, cells were first permeabilized and then stained under similar conditions using the anti-idiotype antibody and anti-EGFR antibody. Expression of the CAR and surrogate marker was analyzed by flow cytometry in CD4+ or CD8+ subsets.

[0318] FIG. 9A and 9B show that the variant CARs containing a modified junction region were expressed on the surface and intracellularly, respectively, in engineered CD4+ T cells. The level of surface expression of the variant CARs was similar or greater than the surface

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[0319] The mean fluorescence intensity (MFI) of CAR surface expression, as determined using the CAR-specific anti-idiotype antibody, was quantified in engineered T cells gated for CD4+ and CD8+ cells. As shown in FIG. 10, surface expression of the CARs was similar on both CD4+ and CD8+ T cells. Variant CARs containing a modified junction region having the L34A and L34S mutations exhibited increased surface expression as compared to the CAR with the native junction region.

B. Cytokine Expression [0320] The production of cytokines by the anti-CD19 CAR T cells, expressing CARs containing either the native junction region or the various modified junction regions, was assessed. Engineered T cells were co-cultured with irradiated CD19-transduced K562 target cells (K562-CD19) or parental K562 cells not expressing the CD 19 antigen in the presence of Golgi inhibitor. After stimulation, the cells were then fixed, permeabilized and intracellular IL2 and IFN-γ cytokine levels were assessed by flow cytometry in CD4+/CAR+ cells and CD8+/CAR+ cells [0321] Exemplary flow cytometry plots depicting intracellular levels of IL-2 and IFN-γ in CD4+/CAR+ cells and CD8+/CAR+ cells are shown in FIG. 11. As shown, a greater percentage of CD4+/CAR+ T cells and CD8+/CAR+ T cells expressing the L34A and L34S variant CARs exhibited intracellular expression of IL-2 after antigen stimulation as compared to cells engineered with the unmodified CAR. A greater percentage of CD8+/CAR+ T cells expressing the R31H and L34A CAR variants had increased IFN-γ cytokine expression after antigen stimulation as compared to cells engineered with the unmodified CAR. Without wishing to be bound by theory, the increased level of cytokine expression may roughly correlate with the higher surface expression observed for the R31H and L34A variant CARs.

C. CAR T Cell Expansion [0322] The ability of cells to expand ex vivo following repeated stimulations in some aspects can indicate capacity of CAR-T cells to persist (e.g. following initial activation) and/or is indicative of function in vivo (Zhao et al. (2015) Cancer Cell, 28:415-28). CAR-T cells were generated as described above and cultured with irradiated target cells (K562-CD19). Cells were stimulated, harvested every 3-4 days and counted, and restimulated with new target cells using the same culture conditions after resetting cell number to initial seeding density for each round.

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A total of 5 rounds of stimulation during a 17 day culture period were carried out. For each round of stimulation, the number of cells was determined.

[0323] As shown in FIG. 12, comparable initial growth of the anti-CD19 CAR-engineered cells was observed for cells expressing variant CARs containing a modified junction region as compared to cells expressing the CAR with the native junction region. After day 7 of restimulation, the degree of cell expansion declined in cells expressing the CAR containing the native junction region, whereas the variant CARs exhibited continued or similar expansion until at least day 10 of restimulation.

[0324] The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any that are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.

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SEQUENCES

Table 5: Sequences

SEQ ID NO	Sequence	Note
1	ESKYGPPCPPCP	IgG4 hinge
2	FWVLVWGGVLACYSLLVTVAFIIFWV	CD28 transmembrane domain
3	KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL	4-IBB costimulatory domain (amino acids 214-255 of Q07011.1) Homo sapien
4	RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR	CD3-zeta intracellular signaling domain
5	FWVLVWGGVLACYSLLVTVAF11FWVKRGRKKLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB
6	CYSLLVTVAFIIFWVKRGRKKLLYIFKQPF	Peptide
7	VAFIIFWVKRGRKKLL	Peptide
8	AFIIFWVKRGRKKLL	Peptide
9	FWVKRGRKKLLYIFK	Peptide
10	FIIFWVKRGRKKLL	Peptide
11	FIIFWVKRGRKKL	Peptide
12	IIFWVKRGRKKLL	Peptide
13	CYSLLVTVAFIIFWVNNKRGRKKLLYIFKQPF	Variant junction region
14	IIFWVNNKRGRKKL	Variant peptide
15	IIFWVNNKRGRKK	Variant peptide
16	VAFIIFWVK	Synthetic peptide
17	AFIIFWVKR	Synthetic peptide
18	FIIFWVKRG	Synthetic peptide
19	IIFWVKRGR	Synthetic peptide
20	IFWVKRGRK	Synthetic peptide
21	FWVKRGRKK	Synthetic peptide
22	WVKRGRKKL	Synthetic peptide
23	VAFIIFWVS	Synthetic peptide K28S
24	AFIIFWVSR	Synthetic peptide K28S
25	FIIFWVSRG	Synthetic peptide K28S
26	IIFWVSRGR	Synthetic peptide K28S
27	IFWVSRGRK	Synthetic peptide K28S
28	FWVSRGRKK	Synthetic peptide K28S
29	WVSRGRKKL	Synthetic peptide K28S
30	VAFIIFWVL	Synthetic peptide

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		K28L
31	AFIIFWVLR	Synthetic peptide K28L
32	FIIFWVLRG	Synthetic peptide K28L
33	IIFWVLRGR	Synthetic peptide K28L
34	IFWVLRGRK	Synthetic peptide K28L
35	FWVLRGRKK	Synthetic peptide K28L
36	WVLRGRKKL	Synthetic peptide K28L
37	VAFIIFWVH	Synthetic peptide K28H
38	AFIIFWVHR	Synthetic peptide K28H
39	FIIFWVHRG	Synthetic peptide K28H
40	IIFWVHRGR	Synthetic peptide K28H
41	IFWVHRGRK	Synthetic peptide K28H
42	FWVHRGRKK	Synthetic peptide K28H
43	WVHRGRKKL	Synthetic peptide K28H
44	VAFIIFWVA	Synthetic peptide K28A
45	AFIIFWVAR	Synthetic peptide K28A
46	IIFWVARGR	Synthetic peptide K28A
47	IFWVARGRK	Synthetic peptide K28A
48	FWVARGRKK	Synthetic peptide K28A
49	WVARGRKKL	Synthetic peptide K28A
50	VAFIIFWVQ	Synthetic peptide K28Q
51	AFIIFWVQR	Synthetic peptide K28Q
52	FIIFWVQRG	Synthetic peptide K28Q
53	IIFWVQRGR	Synthetic peptide K28Q
54	IFWVQRGRK	Synthetic peptide K28Q
55	FWVQRGRKK	Synthetic peptide K28Q
56	WVQRGRKKL	Synthetic peptide

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		K28Q
57	IIFWVKRGS	Synthetic peptide R31S
58	IFWVKRGSK	Synthetic peptide R31S
59	FWVKRGSKK	Synthetic peptide R31S
60	WVKRGSKKL	Synthetic peptide R31S
61	IIFWVKRGL	Synthetic peptide R31L
62	IFWVKRGLK	Synthetic peptide R31L
63	FWVKRGLKK	Synthetic peptide R31L
64	WVKRGLKKL	Synthetic peptide R31L
65	IIFWVKRGH	Synthetic peptide R31H
66	IFWVKRGHK	Synthetic peptide R31H
67	FWVKRGHKK	Synthetic peptide R31H
68	WVKRGHKKL	Synthetic peptide R31H
69	IIFWVKRGA	Synthetic peptide R31A
70	IFWVKRGAK	Synthetic peptide R31A
71	FWVKRGAKK	Synthetic peptide R31A
72	WVKRGAKKL	Synthetic peptide R31A
73	IIFWVKRGN	Synthetic peptide R31N
74	IFWVKRGNK	Synthetic peptide R31N
75	FWVKRGNKK	Synthetic peptide R31N
76	WVKRGNKKL	Synthetic peptide R31N
77	IIFWVQRGS	Synthetic peptide K28Q/R31S
78	IFWVQRGSK	Synthetic peptide K28Q/R31S
79	FWVQRGSKK	Synthetic peptide K28Q/R31S
80	WVQRGSKKL	Synthetic peptide K28Q/R31S
81	IIFWVQRGA	Synthetic peptide K28Q/R31A

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82	IFWVQRGAK	Synthetic peptide K28Q/R31A
83	FWVQRGAKK	Synthetic peptide K28Q/R31A
84	WVQRGAKKL	Synthetic peptide K28Q/R31A
85	IIFWVQRGN	Synthetic peptide K28Q/R31N
86	IFWVQRGNK	Synthetic peptide K28Q/R31N
87	FWVQRGNKK	Synthetic peptide K28Q/R31N
88	WVQRGNKKL	Synthetic peptide K28Q/R31N
89	WVKRGRKKS	Synthetic peptide L34S
90	WVKRGRKKA	Synthetic peptide L34A
91	WVQRGNKKS	Synthetic peptide K28Q/L34S
92	WVQRGNKKA	Synthetic peptide K28Q/L34A
93	MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCP PNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAG CSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVL VNGTKERDWCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTS TALLFLLFFLTLRFSWKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCEL	4-IBB costimulatory domain (Accession No. Q07011.1) Homo sapien
94	MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSR EFRASLHKGLD SAVEVCWYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDI YFCKIEVMYPP PYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLL VTVAFIIFWVR SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS	CD28 transmembrane domain (Accession No. Pl0747) Homo sapien
95	MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTAL FLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST ATKDTYDALHMQALPPR	CD3 zeta chain (Accession No. P20963) Homo sapien
96	FIIFWVNNKRGRKK	Synthetic peptide
97	IFWVNNKRGRKKLL	Synthetic peptide
98	FIIFWVNNKRGRKK	Synthetic peptide
99	IFWVNNKRGRKKLL	Synthetic peptide
100	VAFIIFWVR	Synthetic peptide K28R
101	AFIIFWVRR	Synthetic peptide K28R
102	FIIFWVARG	Synthetic peptide K28A
103	MFWVLVWGGVLACYSLLVTVAFIIFWV	CD28 transmembrane domain (amino acids 153-179 of

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		Accession No. P10747) Homo sapien
104	IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWGGV LACYSLLVTVAFIIFWV	CD28, including transmembrane (amino acids 114-179 of Accession No. P10747) Homo sapien
105	RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR	CD3 zeta Homo sapien
106	GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT	spacer (IgG4hinge) (nt) homo sapien
107	ESKYGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSLGK	Hinge-CH3 spacer Homo sapien
108	ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVS QEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK	Hinge-CH2-CH3 spacer Homo sapien
109	RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKE KEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFWGSD LKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWNAGT SVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLC EVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVP APPSPQPATYTCWSHEDSRTLLNASRSLEVSYVTDH	IgD-hinge-Fc Homo sapien
110	LEGGGEGRGSLLTCGDVEENPGPR	T2A artificial
111	MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFK NCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQA WPENRTDLHAFENLEIIRGRTKQHGQFSLAWSLNITSLGLRSLKEI SD GDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCKATGQVCH ALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECI QCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNT LVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGAL LLLLWALGIGLFM	tEGFR artificial
112	RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS	CD28 cytoplasmic domain (amino acids 180-220 ofP10747) Homo sapien
113	RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS	CD28 cytoplasmic domain variant (LL to GG) Homo sapien
114	FWVLVWGGVLACYSLLVTVAF11FWVARGRKKLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB K28A variant
115	FWVLVWGGVLAC YS LLVTVAF 11FWVHRGRKKLLYI FKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB K28H variant
116	FWVLVWGGVLAC YS LLVTVAF 11FWVLRGRKKLLYI FKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB K28L variant
117	FWVLVWGGVLAC YS LLVTVAF 11FWVQRGRKKLLYI FKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB K28Q variant

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118	FWVLVWGGVLAC YS LLVTVAF11FWVSRGRKKLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB K28S variant
119	FWVLVWGGVLACYSLLVTVAFIIFWVKRGAKKLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB R31A variant
120	FWVLVWGGVLAC YS LLVTVAF 11FWVKRGHKKLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB R31H variant
121	FWVLVWGGVLAC YS LLVTVAF 11FWVKRGLKKLLYI FKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB R31L variant
122	FWVLVWGGVLAC YS LLVTVAF 11FWVKRGNKKLLYI FKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB R31N variant
123	FWVLVWGGVLAC YS LLVTVAF 11FWVKRGRKKALYI FKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB L34A variant
124	FWVLVWGGVLACYSLLVTVAFI IFWVKRGRKKSLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB L34S variant
125	FWVLVWGGVLACYSLLVTVAFI IFWVQRGAKKLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB K28Q/R31A variant
126	FWVLVWGGVLAC YS LLVTVAF 11FWVQRGNKKLLYI FKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB K28Q/R31N variant
127	FWVLVWGGVLACYSLLVTVAFI IFWVQRGSKKLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB K28Q/R31S variant
128	FWVLVWGGVLAC YS LLVTVAF 11FWVQRGRKKALYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB K28Q/L34A variant
129	FWVLVWGGVLACYSLLVTVAFI IFWVQRGRKKSLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB K28Q/L34S variant
130	FWVLVWGGVLAC YS LLVTVAF 11FWVKRGNKKALYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB R31N/L34A variant
131	FWVLVWGGVLACYSLLVTVAFI IFWVKRGNKKSLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB R31N/L34S variant
132	FWVLVWGGVLAC YS LLVTVAF 11FWVQRGNKKALYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB K28Q/R31N/L34A variant
133	FWVLVWGGVLACYSLLVTVAFI IFWVQRGNKKSLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB K28Q/R31N/L34S variant
134	FWVLVWGGVLAC YS LLVTVAF 11FWVNNKRGRKKLL YIFKQPFMRPVQ TTQEEDGCSCRFPEEEEGGCEL	CD28-4-1BB with variant junction region with NN insertion
135	MFWVLVWGGVLAC YS LLVTVAF 11FWVKRGRKKLL YIFKQPFMRPVQT TQEEDGCSCRFPEEEEGGCEL	CD28-4-1BB
136	IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWGGV LACYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF PEEEEGGCEL	CD28-4-1BB
137	S L LVTVAF11FWVKRGRKKLLYIFKQ	CD28-4-1BB junction region
138	S L LVTVAF11FWVARGRKKLLYIFKQ	CD28-4-1BB junction region K14A variant
139	S L LVTVAF11FWVHRGRKKLLYIFKQ	CD28-4-1BB junction region K14H variant

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140	S LLVTVAF11FWVLRGRKKLLYIFKQ	CD28-4-1BB junction region K14L variant
141	S LLVTVAF11FWVQRGRKKLLYIFKQ	CD28-4-1BB junction region K14Q variant
142	S LLVTVAF11FWVSRGRKKLLYIFKQ	CD28-4-1BB junction region K14S variant
143	S L LVTVAF11FWVKRGAKKLLYIFKQ	CD28-4-1BB junction region R17A variant
144	S LLVTVAF11FWVKRGHKKLLYIFKQ	CD28-4-1BB junction region R17H variant
145	S LLVTVAF11FWVKRGLKKLLYIFKQ	CD28-4-1BB junction region R17L variant
146	S L LVTVAF11FWVKRGNKKLLYIFKQ	CD28-4-1BB junction region R17N variant
147	SLLVTVAFIIFWVKRGRKKALYIFKQ	CD28-4-1BB junction region L20A variant
148	S L LVTVAF11FWVKRGRKKS LYIFKQ	CD28-4-1BB junction region L20S variant
149	S L LVTVAF11FWVQRGAKKLLYIFKQ	CD28-4-1BB junction region K14Q/R17A variant
150	S L LVTVAF11FWVQRGNKKLLYIFKQ	CD28-4-1BB junction region K14Q/R17N variant
151	SLLVTVAFIIFWVQRGSKKLLYIFKQ	CD28-4-1BB junction region K14Q/R17S variant
152	SLLVTVAFIIFWVQRGRKKALYIFKQ	CD28-4-1BB junction region K14Q/L20A variant
153	SLLVTVAFIIFWVQRGRKKSLYIFKQ	CD28-4-1BB junction region K14Q/L20S variant
154	SLLVTVAFIIFWVKRGNKKALYIFKQ	CD28-4-1BB junction region R17N/L20A variant
155	SLLVTVAFIIFWVKRGNKKSLYIFKQ	CD28-4-1BB junction region R17N/L20S variant
156	S LLVTVAF11FWVQRGNKKALYIFKQ	CD28-4-1BB junction region K14Q/R17N/L20A variant
157	SLLVTVAFIIFWVQRGNKKSLYIFKQ	CD28-4-1BB junction region K14Q/R17N/L20S variant
158	GAATCTAAGTACGGACCGCCCTGCC	spacer (IgG4hinge)

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	CCCCTTG CCCT	(nucleotide) homo sapien
159	RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK DTYDALHMQALPPR	CD3 Zeta
160	CYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQT	peptide
161	CYSLLVTVAFIIFWV	Synthetic peptide
162	LLVTVAFIIFWVKRG	Synthetic peptide
163	TVAFIIFWVKRGRKK	Synthetic peptide
164	FIIFWVKRGRKKLLY	Synthetic peptide
165	FWVKRGRKKLLYIFK	Synthetic peptide
166	KRGRKKLLYIFKQPF	Synthetic peptide
167	RKKLLYIFKQPFMRP	Synthetic peptide
168	LLYIFKQPFMRPVQT	Synthetic peptide
169	TVAFIIFWVKRGHKK	Synthetic peptide R31H
170	FIIFWVKRGHKKLLY	Synthetic peptide R31H
171	FWVKRGHKKLLYIFK	Synthetic peptide R31H
172	KRGHKKLLYIFKQPF	Synthetic peptide R31H
173	HKKLLYIFKQPFMRP	Synthetic peptide R31H
174	TVAFIIFWVKRGNKK	Synthetic peptide R31N
175	FIIFWVKRGNKKLLY	Synthetic peptide R31N
176	FWVKRGNKKLLYIFK	Synthetic peptide R31N
177	KRGNKKLLYIFKQPF	Synthetic peptide R31N
178	NKKLLYIFKQPFMRP	Synthetic peptide R31N
179	FIIFWVKRGRKKALY	Synthetic peptide L34A
180	FWVKRGRKKALYIFK	Synthetic peptide L34A
181	KRGRKKALYIFKQPF	Synthetic peptide L34A
182	RKKALYIFKQPFMRP	Synthetic peptide L34A
183	FWVLVWGGVLACYSLLVTVAFIIFWVKRGSKKLLYIFKQPFMRPVQTT QEEDGCSCRFPEEEEGGCEL	CD28-4-1BB R31S variant
184	SLLVTVAFIIFWVKRGSKKLLYIFKQ	CD28-4-1BB junction region R31S variant

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Claims

WHAT IS CLAIMED:

1. A variant chimeric receptor, comprising a modified junction region having one or more amino acid sequence modifications compared to a junction region of a reference chimeric receptor, wherein:

the reference chimeric receptor comprises a first domain and a second domain, joined in contiguous sequence at a junction, wherein the junction region of the reference chimeric receptor comprises up to 15 contiguous amino acids directly C-terminal of the junction and/or up to 15 contiguous amino acids directly N-terminal of the junction; and a peptide fragment having the sequence of an 8-15 amino acid portion of the modified junction region has a binding affinity for a human leukocyte antigen (HLA) molecule that is lower than the binding affinity, for the same HLA molecule, of a peptide fragment having the sequence of the corresponding portion of the junction region of the reference chimeric receptor.
2. The variant chimeric receptor of claim 1, wherein the peptide fragment of the corresponding portion of the junction region of the reference chimeric receptor has a binding affinity of less than 1000 nM, less than 500 nM or less than 50 nM.
3. A variant chimeric receptor, comprising a modified junction region having one or more amino acid sequence modifications compared to a junction region of a reference chimeric receptor, wherein:

the reference chimeric receptor comprises a first domain and a second domain, joined in contiguous sequence at a junction, wherein the junction region of the reference chimeric receptor comprises up to 15 contiguous amino acids directly C-terminal of the junction and/or up to 15 contiguous amino acids directly N-terminal of the junction; and the average of the binding affinities of all 8-15 amino acid fragments, or of all 8, 9, 10, 11, 12, 13, 14, or 15 amino acid fragments, within the modified junction region for a human HLA molecule is lower than the average of the binding affinities of all 8-15 amino acid fragments, or of all 8, 9, 10, 11, 12, 13, 14, or 15 amino acid fragments, within the junction region of the reference chimeric receptor.

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4. The variant chimeric receptor of any of claims 1-3, wherein the binding affinity or average of binding affinities is more than 2-fold, more than 5-fold, more than 10-fold, more than 25-fold, more than 50-fold or more than 100-fold lower.
5. A variant chimeric receptor, comprising a modified junction region having one or more amino acid sequence modifications compared to a junction region of a reference chimeric receptor, wherein:

the reference chimeric receptor comprises a first domain directly linked to a second domain joined in contiguous sequence at a junction, wherein;

the junction region of the reference chimeric receptor comprises up to 15 contiguous amino acids directly C-terminal of the junction and/or up to 15 contiguous amino acids directly N-terminal of the junction; and the number of peptide fragments having the sequence of an 8-15 amino acid portion of the modified junction region that has a binding affinity for a human leukocyte antigen (HLA) of less than 1000 nM is reduced compared to the number of peptide fragments having the sequence of an 8-15 amino acid portion of the junction region of the reference chimeric receptor that has the same affinity for binding the same HLA.
6. The variant chimeric receptor of claim 5, wherein:

the number of peptide fragments within the modified junction region that exhibits a binding affinity for an HLA of less than 500 nM or less than 50 nM is reduced; or the binding affinity of less than 1000 nM is a binding affinity of less than 500 nM or less than 50 nm.
7. The variant chimeric receptor of any of claims 1-6, wherein the binding affinity is an IC50 and the comparison of binding of peptide fragments of the modified junction regions and the peptide fragments of the junction region of the reference chimeric receptor is with reference to the same standard peptide.
8. The variant chimeric receptor of any of claims 1-7, wherein:

the first domain and/or second domain comprises a domain of a natural human protein;

and/or

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9. The variant chimeric receptor of any of claims 1-8, wherein the first domain and second domain are not present in the same molecule in vivo in a human subject.
10. The variant chimeric receptor of any of claims 1-9, wherein the first domain and second domain are, respectively, an extracellular ligand binding domain and a hinge domain, a hinge domain and a transmembrane domain, a transmembrane domain and an intracellular costimulatory signaling domain, and an intracellular costimulatory signaling domain and an activating cytoplasmic signaling domain, which can include functional portions of such domains.
11. The variant chimeric receptor of any of claims 1-10, wherein the first domain is a transmembrane domain or a functional portion thereof and the second domain is a costimulatory signaling domain or a functional portion thereof.
12. The variant chimeric receptor of claim 11, wherein the transmembrane domain is a CD28 transmembrane domain or a functional portion or variant thereof and the costimulatory signaling domain is a 4-IBB signaling domain or a functional portion or variant thereof.
13. The variant chimeric receptor of any of claims 1-12, wherein the junction region of the reference chimeric receptor comprises up to 13 contiguous amino acids directly Cterminal of the junction and/or up to 15 contiguous amino acids directly N-terminal of the junction.
14. The variant chimeric receptor of any of claims 1-13, wherein the peptide fragment(s) comprises a sequence of amino acids between or between about 8 and 15 amino acids in length, or comprises a sequence of amino acids that is at least or at least about or is or is about 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in length.

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15. The variant chimeric receptor of any of claims 1-14, wherein the variant chimeric receptor comprises:

a domain of at least 95% sequence identity to the first domain and/or a domain of at least 95% sequence identity to the second domain;

a domain identical in sequence to the first domain and a domain of at least 95% sequence identity to the second domain; or a domain of at least 95% sequence identity to the first domain and a domain identical in sequence to the second domain, wherein at least one or both of the domains present in the variant chimeric receptor is modified compared to the first domain and/or second domain of the reference chimeric receptor in the portion comprising the modified junction region.
16. The variant chimeric receptor of any of claims 1-15, wherein:

the variant chimeric receptor comprises at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference chimeric receptor; and/or the variant chimeric receptor comprises up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,

15, 16, 17, 18, 19 or 20 amino acid modifications compared to the reference chimeric receptor.
17. The variant chimeric receptor of any of claims 12-16, wherein the CD28 transmembrane domain comprises the sequence of amino acids set forth in SEQ ID NO:2, 103 or 104 or a functional portion or variant thereof comprising a sequence that exhibits at least 95% sequence identity to SEQ ID NO:2, 103 or 104; and the 4-IBB costimulatory signaling domain comprises the sequence of amino acids set forth in SEQ ID NO:3 or a functional portion or variant thereof comprising a sequence that exhibits at least 95% sequence identity to SEQ ID NO:3.
18. The variant chimeric receptor of any of claims 12-17, wherein the first domain and second domain together comprise the sequence of amino acids set forth in SEQ ID NO:5 or a functional portion or variant thereof comprising a sequence of amino acids that exhibits at least

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85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:5.
19. The variant chimeric receptor of any of claims 12-18, wherein the first domain and second domain together comprise the sequence of amino acids set forth in SEQ ID NO:5.
20. The variant chimeric receptor of any of claims 12-19, wherein the one or more modifications are within a portion between residue 13 and 42 or between amino acid residue 15 and 40, with reference to numbering set forth in SEQ ID NO:5.
21. The variant chimeric receptor of any of claims 1-20, wherein the one or more modifications comprises an amino acid insertion, replacement or deletion and/or wherein each of the one or more modifications individually comprises an amino acid insertion, replacement, or deletion.
22. The variant chimeric receptor of any of claims 1-21, wherein the first and/or second domain comprises a transmembrane domain, wherein the one or more modifications is not or does not comprise a modification at or of a hydrophobic amino acid residue or within a hydrophobic portion in the transmembrane domain, which optionally is the CD28 transmembrane domain; or the one or more modifications comprises a modification of a hydrophobic amino acid residue within the transmembrane domain, which optionally is the CD28 transmembrane domain, wherein the modification is or comprises a substitution of the hydrophobic amino acid with another different hydrophobic amino acid residue; or the one or more modifications is not or does not comprise a modification at or of a hydrophobic amino acid residue or within a hydrophobic portion in the transmembrane domain, which optionally is the CD28 transmembrane domain, other than a substitution with another hydrophobic amino acid residue.
23. The variant chimeric receptor of any of claims 12-22, wherein the one or more modifications comprise a modification at an amino acid residue corresponding to an amino acid residue between residues 28 and 42 with reference to numbering set forth in SEQ ID NO:5.

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24. The variant chimeric receptor of any of claims 1-23, wherein the amino acid modification is an insertion and the variant chimeric receptor comprises an insertion between amino acid residues adjacent to the junction between the domains, which optionally corresponds to amino acid residues 27 and 28 with reference to numbering set forth SEQ ID NO:5.
25. The variant chimeric receptor of claim 24, comprising insertion of 1, 2, 3, 4 or 5 amino acid residues.
26. The variant chimeric receptor of claim 24 or claim 25, wherein the insertion is of any amino acid residue, which, optionally, is asparagine (N).
27. The variant chimeric receptor of any of claims 12-26, wherein the one or more modifications comprises an amino acid replacement(s) and the amino acid replacement(s) are at one or more residues corresponding to a residue selected from 28, 31 or 34 with reference to numbering set forth in SEQ ID NO:5.
28. The variant chimeric receptor of claim 27, wherein the amino acid replacement(s) is to any other amino acid residue, which, optionally, is selected from among leucine (L), asparagine (N), glutamine (Q), alanine (A), serine (S) or histidine (H).
29. The variant chimeric receptor of claim 27 or claim 28, wherein the amino acid replacement(s) corresponds to or is a replacement selected from among K28A, K28H, K28L, K28Q, K28S, R31A, R31H, R31L, R31N, R31S, L34A and L34S, with reference to numbering set forth in SEQ ID NO:5.
30. The variant chimeric receptor of any of claims 27-29, wherein the amino acid replacement does not comprise the single amino acid replacement corresponding to L34A or L34S, with reference to numbering set forth in SEQ ID NO:5.

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31. The variant chimeric receptor of any of claims 27-30, wherein the amino acid replacements are or correspond to amino acid replacements selected from among K28Q/R31A, K28Q/R31N, K28Q/R31S, K28Q/L34A, K28Q/L34S, R31N/L34A, R31N/L34S,

K28Q/R31N/L34A, K28Q/R31N/L34S.
32. The variant chimeric receptor of any of claims 1-31, wherein:

the variant chimeric receptor comprises a modified junction region comprising less than 100% sequence identity to SEQ ID NO: 137 but greater than 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96% to SEQ ID NO: 137 and comprises the modifications; and/or the variant chimeric receptor comprises a sequence comprising less than 100% sequence identity to SEQ ID NO:5 but greater than 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or 96% to SEQ ID NO:5 and comprises the modifications.
33. The variant chimeric receptor of any of claims 1-32, wherein the variant chimeric receptor comprises a modified junction region selected from among:

i) the sequence of amino acids set forth in any of SEQ ID NOS: 138-157 and 184;

ii) a functional variant thereof comprising a sequence of amino acids that exhibits at least 95% sequence identity to the sequence of amino acids set forth in any of SEQ ID NOS: 138-157 and 184 and that comprise the modification(s); or iii) a functional portion of i) or ii) and comprises the modification(s).
34. The variant chimeric receptor of any of claims 1-33, wherein the variant chimeric receptor comprises:

i) the sequence of amino acids set forth in any of SEQ ID NOS: 114-134 and 183;

ii) a functional variant thereof comprising a sequence of amino acids that exhibits at least 95% sequence identity to the sequence of amino acids set forth in any of SEQ ID NOS: 114-134 and 183 and that comprise the modification(s);

ii) or is a functional portion of i) or ii) and comprises the modification(s).
35. The variant chimeric receptor of any of claims 1-34, wherein the modified junction region comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications compared to the junction region of the reference chimeric receptor.

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36. The variant chimeric receptor of any of claims 1-35, wherein the first domain or second domain is a transmembrane domain and the corresponding domain in the variant chimeric receptor comprises a substantially hydrophobic hydropathy profile and/or comprises a grand average of hydropathy (GRAVY) value of greater than 0, 0.25, 0.5, 0.75, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2 or greater.
37. The variant chimeric receptor of any of claims 1-36, wherein first domain or second domain comprises an intracellular signaling domain and the corresponding domain in the variant chimeric receptor is capable of inducing the activation or cellular localization of a TRAF and/or is capable of inducing TRAF-mediated signaling.
38. The variant chimeric receptor of claim 37, wherein the intracellular signaling domain is a 4-IBB co-stimulatory signaling domain and/or the TRAF is selected from among TRAF1, TRAF2 or TRAF3.
39. The variant chimeric receptor of any of claims 12-38, wherein the variant chimeric receptor comprises amino acids TTQE at positions corresponding to 49-52 and/or amino acids PEEE at positions corresponding to residues 60-63, each with reference to numbering set forth in SEQ ID NO:5.
40. The variant chimeric receptor of any of claims 1-39, wherein the HLA is an HLA class I and/or an HLA class II.
41. The variant chimeric receptor of any of claims 1-40, wherein the HLA class I is selected from an HLA allele set forth in Table 1A and/or the HLA class II allele is selected from an HLA allele set forth in Table IB.
42. The variant chimeric receptor of any of claims 1-41, wherein the HLA class I allele is selected from among HLA-A*02:01, HLA-A*03:01, HLA-A*ll:01 and HLA-B*08:01.

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43. The variant chimeric receptor of any of claims 3-42, wherein the HLA comprises a plurality of HLA molecules and average binding affinities to one or more of the plurality of HLA molecules is lower and/or the number of peptide fragments that individually bind to one or more of the plurality of HLA molecules is reduced.
44. The variant chimeric receptor of claim 43, wherein the plurality of HLA molecules is selected from:

a plurality of HLA class I molecules that represent greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95% or greater than 99% of the HLA class I molecules in the worldwide population or in the Caucasian population;

a plurality of HLA class II molecules that represent greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95% or greater than 99% of the HLA class II molecules in the worldwide population or in the Caucasian population; or a plurality of HLA class I molecules and HLA class II molecules that represent greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95% or greater than 99% of the HLA class I molecules and HLA class II molecules in the worldwide population or in the Caucasian population;
45. The variant chimeric receptor of any of claims 1-44, wherein the binding affinity is as determined in vitro.
46. The variant chimeric receptor of any of claims 1-45 that exhibits reduced immunogenicity compared to the reference chimeric receptor upon administration to a human subject, optionally wherein the subject has received administration of the reference chimeric receptor.
47. The variant chimeric receptor of claim 46, wherein the reduced immunogenicity comprises a reduced CD4+ T cell immune response and/or a reduced CD8+ T cell immune response.

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48. The variant chimeric receptor of any of claims 11-47, wherein:

the reference chimeric receptor further comprises an extracellular ligand-binding domain; and/or the variant chimeric receptor further comprises an extracellular ligand-binding domain.
49. The variant chimeric receptor of claim 48 that is a chimeric antigen receptor (CAR), wherein the ligand-binding domain is an antigen-binding domain.
50. The variant chimeric receptor of claim 49, wherein the antigen-binding domain is an antibody or an antibody fragment.
51. The variant chimeric receptor of claim 50, wherein the antigen-binding domain is an antibody fragment that is a single chain fragment.
52. The variant chimeric receptor of claim 50 or claim 51, wherein the fragment comprises antibody variable regions joined by a flexible immunoglobulin linker.
53. The variant chimeric receptor of any of claims 50-52, wherein the antibody fragment comprises an scFv.
54. The variant chimeric receptor of any of claims 48-52, wherein the ligand-binding domain specifically binds an antigen that is associated with a disease or disorder.
55. The variant chimeric receptor of claim 54, wherein:

the disease or disorder is an infectious disease or condition, an autoimmune disease, an inflammatory disease or a tumor or a cancer;

the ligand-binding domain specifically binds to a tumor antigen; and/or the ligand-binding domain specifically binds to an antigen selected from the group consisting of ROR1, Her2, Ll-CAM, CD19, CD20, CD22, mesothelin, CEA, hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, ErbB3, ErbB4, FBP, fetal acethycholine e receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, Ll-cell adhesion molecule,

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MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1, gplOO, oncofetal antigen, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, estrogen receptor, progesterone receptor, ephrinB2, CD123, CS-1, cMet, GD-2, MAGE A3, CE7, Wilms Tumor 1 (WT-1) and cyclin Al (CCNA1).
56. The variant chimeric receptor of any of claims 11-55, wherein:

the reference chimeric receptor further comprises an activating cytoplasmic signaling domain; and/or the variant chimeric receptor further comprises an activating cytoplasmic domain.
57. The variant chimeric receptor of claim 56, wherein the activating cytoplasmic domain comprises a T cell receptor (TCR) component and/or comprise an immunoreceptor tyrosine-based activation motif (ΓΓΑΜ).
58. The variant chimeric receptor of claim 56 or claim 57, wherein the activating cytoplasmic signaling domain is or comprises a cytoplasmic signaling domain of a zeta chain of a CD3-zeta (Οϋ3ζ) chain or a functional variant or signaling portion thereof.
59. The variant chimeric receptor of any of claims 11-58, wherein:

the reference chimeric receptor comprises form its N to C terminus in order: an extracellular ligand-binding domain, the first domain that is a transmembrane domain, the second domain that is an intracellular costimulatory domain and an activating cytoplasmic signaling domain; and/or the variant chimeric receptor comprises from its N to C terminus in order: an extracellular ligand-binding domain, a transmembrane domain, a intracellular costimulatory domain and an activating cytoplasmic signaling domain, wherein the transmembrane domain and intracellular costimulatory domain are joined in contiguous sequence at a junction to form the modified junction region.
60. A nucleic acid molecule encoding the variant chimeric receptor of any of claims

1-59.

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61. A vector, comprising the nucleic acid molecule of claim 60.
62. The vector of claim 61 that is a viral vector.
63. The vector of claim 61 or claim 62 that is a retroviral vector, which optionally is a lentiviral vector or a gammaretroviral vector.
64. An engineered cell, comprising the nucleic acid of claim 60 or the vector of any of claims 61-63 or expressing the chimeric receptor of any of claims 1-59.
65. The engineered cell of claim 64, which is a T cell.
66. The engineered cell of claim 64 or claim 65 that is a CD4+ T cell and/or a CD8+

T cell.
67. A composition, comprising the engineered cells of any of claims 64-66, and optionally a pharmaceutically acceptable buffer.
68. A method of treatment, comprising administering the cell of any of claims 64-66 or the composition of claim 67 to a subject having a disease or condition.
69. The method of claim 68, wherein the chimeric receptor specifically binds to a ligand or antigen associated with the disease or condition.
70. The method of claim 68 or claim 69, wherein the disease or condition is a cancer, a tumor, an autoimmune disease or disorder, or an infectious disease.
71. The method of any of claims 68-70, wherein the genetically engineered T cells in the composition exhibit increased or longer expansion and/or persistence in the subject than in a subject administered the same or about the same dosage amount of a reference cell composition expressing the reference chimeric receptor.

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72. The method of claim 71, wherein the increase is at least 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, or 5-fold.

73. The method of claim 71 or claim 72, wherein the increase is observed or is present within a month, within two months, within six months or within one year of administering the cells.

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735042004740SEQLIST <210> 12 <211> 13 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <400> 12

Ile Ile Phe Trp Val Lys Arg Gly Arg Lys Lys Leu Leu 1 5 10 <210> 13 <211> 32 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> Variant junction region <400> 13

Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Asn 1 5 10 15 Asn Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 20 25 30

<210> 14 <211> 14 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> Variant peptide <400> 14

Ile Ile Phe Trp Val Asn Asn Lys Arg Gly Arg Lys Lys Leu 1 5 10 <210> 15 <211> 13 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> Variant peptide

<400> 15 Ile Ile Phe Trp Val Asn Asn Lys Arg Gly Arg Lys Lys 1 5 10 <210> <211> <212> <213> 16 9 PRT Artificial Sequence

Page 4

735042004740SEQLIST <220>

<223> Synthetic <400> 16

Val Ala Phe Ile Ile Phe Trp Val Lys 1 5 <210> 17 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <400> 17

Ala Phe Ile Ile Phe Trp Val Lys Arg 1 5 <210> 18 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <400> 18

Phe Ile Ile Phe Trp Val Lys Arg Gly 1 5 <210> 19 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <400> 19

Ile Ile Phe Trp Val Lys Arg Gly Arg 1 5 <210> 20 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <400> 20

Ile Phe Trp Val Lys Arg Gly Arg Lys 1 5 <210> 21 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic

Page 5

735042004740SEQLIST <400> 21

Phe Trp Val Lys Arg Gly Arg Lys Lys 1 5 <210> 22 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <400> 22

Trp Val Lys Arg Gly Arg Lys Lys Leu 1 5 <210> 23 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28S <400> 23

Val Ala Phe Ile Ile Phe Trp Val Ser 1 5 <210> 24 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28S <400> 24

Ala Phe Ile Ile Phe Trp Val Ser Arg 1 5 <210> 25 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28S <400> 25

Phe Ile Ile Phe Trp Val Ser Arg Gly

1 5 <210> 26 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide K28S

Page 6

735042004740SEQLIST <400> 26

Ile Ile Phe Trp Val Ser Arg Gly Arg 1 5 <210> 27 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28S <400> 27

Ile Phe Trp Val Ser Arg Gly Arg Lys 1 5 <210> 28 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28S <400> 28

Phe Trp Val Ser Arg Gly Arg Lys Lys 1 5 <210> 29 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28S <400> 29

Trp Val Ser Arg Gly Arg Lys Lys Leu 1 5 <210> 30 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28L <400> 30

Val Ala Phe Ile Ile Phe Trp Val Leu 1 5 <210> 31 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28L <400> 31

Ala Phe Ile Ile Phe Trp Val Leu Arg

Page 7

735042004740SEQLIST <210> 32 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28L <400> 32

Phe Ile Ile Phe Trp Val Leu Arg Gly 1 5 <210> 33 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28L <400> 33

Ile Ile Phe Trp Val Leu Arg Gly Arg 1 5 <210> 34 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28L <400> 34

Ile Phe Trp Val Leu Arg Gly Arg Lys 1 5 <210> 35 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28L <400> 35

Phe Trp Val Leu Arg Gly Arg Lys Lys 1 5 <210> 36 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28L <400> 36

Trp Val Leu Arg Gly Arg Lys Lys Leu 1 5

Page 8

735042004740SEQLIST <210> 37 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28H <400> 37

Val Ala Phe Ile Ile Phe Trp Val His 1 5 <210> 38 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28H <400> 38

Ala Phe Ile Ile Phe Trp Val His Arg 1 5 <210> 39 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28H <400> 39

Phe Ile Ile Phe Trp Val His Arg Gly 1 5 <210> 40 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28H <400> 40

Ile Ile Phe Trp Val His Arg Gly Arg 1 5 <210> 41 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28H <400> 41

Ile Phe Trp Val His Arg Gly Arg Lys 1 5 <210> 42 <211> 9 <212> PRT

Page 9

735042004740SEQLIST <213> Artificial Sequence <220>

<223> Synthetic peptide K28H <400> 42

Phe Trp Val His Arg Gly Arg Lys Lys 1 5 <210> 43 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28H <400> 43

Trp Val His Arg Gly Arg Lys Lys Leu 1 5 <210> 44 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28A <400> 44

Val Ala Phe Ile Ile Phe Trp Val Ala 1 5 <210> 45 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28A <400> 45

Ala Phe Ile Ile Phe Trp Val Ala Arg 1 5 <210> 46 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28A <400> 46

Ile Ile Phe Trp Val Ala Arg Gly Arg 1 5 <210> 47 <211> 9 <212> PRT <213> Artificial Sequence <220>

Page 10

735042004740SEQLIST <223> Synthetic peptide K28A <400> 47

Ile Phe Trp Val Ala Arg Gly Arg Lys 1 5 <210> 48 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28A <400> 48

Phe Trp Val Ala Arg Gly Arg Lys Lys 1 5 <210> 49 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28A <400> 49

Trp Val Ala Arg Gly Arg Lys Lys Leu 1 5 <210> 50 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28Q <400> 50

Val Ala Phe Ile Ile Phe Trp Val Gln 1 5 <210> 51 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28Q <400> 51

Ala Phe Ile Ile Phe Trp Val Gln Arg 1 5 <210> 52 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28Q <400> 52

Page 11

735042004740SEQLIST Phe Ile Ile Phe Trp Val Gln Arg Gly

1 5 <210> 53 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28Q <400> 53

Ile Ile Phe Trp Val Gln Arg Gly Arg 1 5 <210> 54 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28Q <400> 54

Ile Phe Trp Val Gln Arg Gly Arg Lys 1 5 <210> 55 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28Q <400> 55

Phe Trp Val Gln Arg Gly Arg Lys Lys 1 5 <210> 56 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28Q <400> 56

Trp Val Gln Arg Gly Arg Lys Lys Leu 1 5 <210> 57 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31S <400> 57

Ile Ile Phe Trp Val Lys Arg Gly Ser 1 5

Page 12

735042004740SEQLIST <210> 58 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31S <400> 58

Ile Phe Trp Val Lys Arg Gly Ser Lys 1 5 <210> 59 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31S <400> 59

Phe Trp Val Lys Arg Gly Ser Lys Lys 1 5 <210> 60 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31S <400> 60

Trp Val Lys Arg Gly Ser Lys Lys Leu 1 5 <210> 61 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31L <400> 61

Ile Ile Phe Trp Val Lys Arg Gly Leu 1 5 <210> 62 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31L <400> 62

Ile Phe Trp Val Lys Arg Gly Leu Lys 1 5 <210> 63 <211> 9

Page 13

735042004740SEQLIST <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31L <400> 63

Phe Trp Val Lys Arg Gly Leu Lys Lys 1 5 <210> 64 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31L <400> 64

Trp Val Lys Arg Gly Leu Lys Lys Leu 1 5 <210> 65 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31H <400> 65

Ile Ile Phe Trp Val Lys Arg Gly His 1 5 <210> 66 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31H <400> 66

Ile Phe Trp Val Lys Arg Gly His Lys 1 5 <210> 67 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31H <400> 67

Phe Trp Val Lys Arg Gly His Lys Lys 1 5 <210> 68 <211> 9 <212> PRT <213> Artificial Sequence

Page 14

735042004740SEQLIST <220>

<223> Synthetic peptide R31H <400> 68

Trp Val Lys Arg Gly His Lys Lys Leu 1 5 <210> 69 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31A <400> 69

Ile Ile Phe Trp Val Lys Arg Gly Ala 1 5 <210> 70 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31A <400> 70

Ile Phe Trp Val Lys Arg Gly Ala Lys 1 5 <210> 71 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31A <400> 71

Phe Trp Val Lys Arg Gly Ala Lys Lys 1 5 <210> 72 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31A <400> 72

Trp Val Lys Arg Gly Ala Lys Lys Leu 1 5 <210> 73 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31N

Page 15

735042004740SEQLIST <400> 73

Ile Ile Phe Trp Val Lys Arg Gly Asn 1 5 <210> 74 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31N <400> 74

Ile Phe Trp Val Lys Arg Gly Asn Lys 1 5 <210> 75 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31N <400> 75

Phe Trp Val Lys Arg Gly Asn Lys Lys 1 5 <210> 76 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31N <400> 76

Trp Val Lys Arg Gly Asn Lys Lys Leu 1 5 <210> 77 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28Q/R31S <400> 77

Ile Ile Phe Trp Val Gln Arg Gly Ser 1 5 <210> 78 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28Q/R31S <400> 78

Ile Phe Trp Val Gln Arg Gly Ser Lys 1 5

Page 16

735042004740SEQLIST <210> 79 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28Q/R31S <400> 79

Phe Trp Val Gln Arg Gly Ser Lys Lys 1 5 <210> 80 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28Q/R31S <400> 80

Trp Val Gln Arg Gly Ser Lys Lys Leu 1 5 <210> 81 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28Q/R31A <400> 81

Ile Ile Phe Trp Val Gln Arg Gly Ala 1 5 <210> 82 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28Q/R31A <400> 82

Ile Phe Trp Val Gln Arg Gly Ala Lys 1 5 <210> 83 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28Q/R31A <400> 83

Phe Trp Val Gln Arg Gly Ala Lys Lys 1 5 <210> 84

Page 17

735042004740SEQLIST <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28Q/R31A <400> 84

Trp Val Gln Arg Gly Ala Lys Lys Leu 1 5 <210> 85 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28Q/R31N <400> 85

Ile Ile Phe Trp Val Gln Arg Gly Asn 1 5 <210> 86 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28Q/R31N <400> 86

Ile Phe Trp Val Gln Arg Gly Asn Lys 1 5 <210> 87 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28Q/R31N <400> 87

Phe Trp Val Gln Arg Gly Asn Lys Lys 1 5 <210> 88 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28Q/R31N <400> 88

Trp Val Gln Arg Gly Asn Lys Lys Leu 1 5 <210> 89 <211> 9 <212> PRT <213> Artificial Sequence

Page 18

735042004740SEQLIST <220>

<223> Synthetic peptide L34S

<400> 89 Trp Val Lys Arg Gly Arg Lys Lys Ser 1 5 <210> 90 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide L34A <400> 90 Trp Val Lys Arg Gly Arg Lys Lys Ala 1 5 <210> 91 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide K28Q/L34S <400> 91 Trp Val Gln Arg Gly Asn Lys Lys Ser 1 5 <210> 92 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide K28Q/L34A <400> 92 Trp Val Gln Arg Gly Asn Lys Lys Ala 1 5 <210> 93 <211> 255 <212> PRT

<213> Homo sapiens <220>

<223> 4-1BB <400> 93

Met Gly Asn 1 Ser Cys 5 Tyr Asn Ile Val Ala 10 Thr Leu Leu Leu Val 15 Leu Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro 20 25 30 Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys 35 40 45 Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile 50 55 60 Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser 65 70 75 80 Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly

Page 19

735042004740SEQLIST

85 90 95 Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu 100 105 110 Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln 115 120 125 Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys 130 135 140 Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro 145 150 155 160 Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala 165 170 175 Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu 180 185 190 Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu 195 200 205 Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 210 215 220 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 225 230 235 240 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 245 250 255

<210> 94 <211> 255 <212> PRT <213> Homo sapiens <220>

<223> CD28 <400> 94

Met 1 Gly Asn Ser Cys Tyr 5 Asn Ile Val Ala 10 Thr Leu Leu Leu Val 15 Leu Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro 20 25 30 Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys 35 40 45 Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile 50 55 60 Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser 65 70 75 80 Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly 85 90 95 Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu 100 105 110 Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln 115 120 125 Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys 130 135 140 Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro 145 150 155 160 Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala 165 170 175 Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu 180 185 190 Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu 195 200 205 Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 210 215 220 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 225 230 235 240 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 245 250 255

<210> 95 <211> 164

Page 20

735042004740SEQLIST <212> PRT <213> Homo sapiens <220>

<223> CD3 zeta chain <400> 95

Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ile Leu Gln Ala Gln Leu 1 5 10 15 Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp Pro Lys Leu Cys 20 25 30 Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val Ile Leu Thr Ala 35 40 45 Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr 50 55 60 Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg 65 70 75 80 Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met 85 90 95 Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 100 105 110 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 115 120 125 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 130 135 140 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 145 150 155 160 Leu Pro Pro Arg

<210> 96 <211> 14 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <400> 96

Phe Ile Ile Phe Trp Val Asn Asn Lys Arg Gly Arg Lys Lys 1 5 10 <210> 97 <211> 14 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <400> 97

Ile Phe Trp Val Asn Asn Lys Arg Gly Arg Lys Lys Leu Leu 1 5 10 <210> 98 <211> 14 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <400> 98

Phe Ile Ile Phe Trp Val Asn Asn Lys Arg Gly Arg Lys Lys 1 5 10 Page 21

735042004740SEQLIST

<210> 99 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 99 Ile Phe Trp Val Asn Asn Lys Arg Gly Arg Lys Lys Leu Leu 1 5 10 <210> 100 <211> 9 <212> PRT <213> Artificial Sequence

<220>

<223> Synthetic peptide K28R <400> 100

Val Ala Phe Ile Ile Phe Trp Val Arg 1 5 <210> 101 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28R <400> 101

Ala Phe Ile Ile Phe Trp Val Arg Arg 1 5 <210> 102 <211> 9 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide K28A <400> 102

Phe Ile Ile Phe Trp Val Ala Arg Gly 1 5 <210> 103 <211> 28 <212> PRT <213> Homo sapiens <220>

<223> CD28 transmembrane domain <400> 103

Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 1 5 10 15 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25

Page 22

735042004740SEQLIST <210> 104 <211> 66 <212> PRT <213> Homo sapiens <220>

<223> CD28 <400> 104

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn 1 5 10 15 Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu 20 25 30 Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly 35 40 45 Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 50 55 60

Trp Val 65 <210> 105 <211> 112 <212> PRT <213> Homo sapiens <220>

<223> CD3 zeta <400> 105

Arg 1 Val Lys Phe Ser Arg Ser Ala 5 Glu Pro 10 Pro Ala Tyr Gln Gln Gly 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110

<210> 106 <211> 36 <212> DNA <213> Homo sapiens <220>

<223> spacer (IgG4hinge) <400> 106 gaatctaagt acggaccgcc ctgcccccct tgccct <210> 107 <211> 119 <212> PRT <213> Homo sapiens <220>

<223> Hinge-CH3 spacer <400> 107

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg Page 23

735042004740SEQLIST

1 5 10 15 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 20 25 30 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 35 40 45 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 50 55 60 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 65 70 75 80 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 85 90 95 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 100 105 110 Leu Ser Leu Ser Leu Gly Lys

115 <210> 108 <211> 229 <212> PRT <213> Homo sapiens <220>

<223> H nge CH2 CH3 spacer <400> 108 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225

<210> 109 <211> 282 <212> PRT <213> Homo sapiens <220> <223> IgD-hinge-Fc

<400> 109

Arg Trp Pro Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro Thr Ala Page 24

735042004740SEQLIST

1 5 10 15

Gln Pro Gln Ala 20 Glu Gly Ser Leu Thr Thr Arg 35 Asn Thr Gly Arg Gly 40 Glu Lys 50 Glu Glu Gln Glu Glu 55 Arg Ser 65 His Thr Gln Pro Leu 70 Gly Val Asp Leu Trp Leu Arg 85 Asp Lys Ala Ser Asp Leu Lys 100 Asp Ala His Leu Pro Thr Gly 115 Gly Val Glu Glu Gly 120 Ser Gln 130 Ser Gln His Ser Arg 135 Leu Ala 145 Gly Thr Ser Val Thr 150 Cys Thr Gln Arg Leu Met Ala 165 Leu Arg Glu Leu Ser Leu Asn 180 Leu Leu Ala Ser Trp Leu Leu 195 Cys Glu Val Ser Gly 200 Met Trp 210 Leu Glu Asp Gln Arg 215 Glu Ala 225 Arg Pro Pro Pro Gln 230 Pro Gly Val Leu Arg Val Pro 245 Ala Pro Pro Cys Val Val Ser 260 His Glu Asp Ser Ser Leu Glu 275 Val Ser Tyr Val Thr 280

Ala Lys Ala Thr Thr Ala Pro Ala 25 Gly Glu Glu Lys Lys 30 Lys Glu Lys Glu Thr Lys Thr 45 Pro Glu Cys Pro Tyr Leu Leu 60 Thr Pro Ala Val Gln Thr Phe 75 Thr Cys Phe Val Val 80 Gly Thr 90 Trp Glu Val Ala Gly 95 Lys Val 105 Leu Leu Glu Arg His 110 Ser Asn Gly Thr Leu Pro Arg 125 Ser Leu Trp Asn Leu Asn His 140 Pro Ser Leu Pro Pro Pro Ala 155 Ala Gln Ala Pro Val 160 Lys Ser 170 Asp Pro Pro Glu Ala 175 Ala Ser 185 Phe Ser Pro Pro Asn 190 Ile Leu Leu Val Asn Thr Ser 205 Gly Phe Ala Pro Ser Thr Thr 220 Phe Trp Ala Trp Ser Ser Pro 235 Gln Pro Ala Thr Tyr 240 Thr Arg 250 Thr Leu Leu Asn Ala 255 Ser Arg 265 Asp His 270

<210> 110 <211> 24 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> T2A <400> 110

Leu 1 Glu Gly Gly Gly 5 Glu Gly Arg Val Glu Glu Asn 20 Pro Gly Pro Arg

Gly Ser Leu Leu Thr Cys Gly Asp 10 15 <210> 111 <211> 357 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> tEGFR <400> 111

Met Leu Leu Leu Val Thr Ser Leu

Leu Leu Cys Glu Leu Pro His Pro Page 25

735042004740SEQLIST

1 5 10 15 Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly 20 25 30 Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe 35 40 45 Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala 50 55 60 Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu 65 70 75 80 Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile 85 90 95 Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu 100 105 110 Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala 115 120 125 Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu 130 135 140 Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr 145 150 155 160 Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys 165 170 175 Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly 180 185 190 Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu 195 200 205 Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys 210 215 220 Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu 225 230 235 240 Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met 245 250 255 Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala 260 265 270 His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val 275 280 285 Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His 290 295 300 Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro 305 310 315 320 Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala 325 330 335 Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly 340 345 350 Ile Gly Leu Phe Met

355 <210> 112 <211> 41 <212> PRT <213> Homo sapiens <220>

<223> CD28 cytoplasmic domain <400> 112

Arg 1 Ser Lys Arg Ser 5 Arg Leu Leu His Ser Asp 10 Tyr Met Asn Met 15 Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40

<210> 113 <211> 41 <212> PRT <213> Homo sapiens

Page 26

735042004740SEQLIST <220>

<223> CD28 cytoplasmic domain <400> 113

Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 114 <211> 69 <212> PRT <213> Artificial Sequence <220> <223> Synthetic K28A <400> 114 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Ala Arg Gly Arg Lys 20 25 30 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 35 40 45 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 50 55 60 Gly Gly Cys Glu 65 Leu <210> 115 <211> 69 <212> PRT <213> Artificial Sequence <220> <223> Synthetic K28H <400> 115 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val His Arg Gly Arg Lys 20 25 30 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 35 40 45 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 50 55 60 Gly Gly Cys Glu 65 Leu <210> 116 <211> 69 <212> PRT <213> Artificial Sequence <220> <223> Synthetic K28L <400> 116 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Leu Arg Gly Arg Lys 20 25 30

Page 27

735042004740SEQLIST

Lys Leu Leu 35 Tyr Ile Phe Lys Gln 40 Pro Phe Met Arg Pro 45 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 50 55 60 Gly Gly Cys Glu Leu

<210> 117 <211> 69 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic K28Q <400> 117

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Gln Arg Gly Arg Lys 20 25 30 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 35 40 45 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 50 55 60 Gly Gly Cys Glu Leu

<210> 118 <211> 69 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic K28S <400> 118

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Ser Arg Gly Arg Lys 20 25 30 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 35 40 45 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 50 55 60 Gly Gly Cys Glu Leu

<210> 119 <211> 69 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic R31A <400> 119

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Ala Lys 20 25 30 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 35 40 45 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 50 55 60 Gly Gly Cys Glu Leu

Page 28

735042004740SEQLIST <210> 120 <211> 69 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic R31H <400> 120

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly His Lys 20 25 30 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 35 40 45 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 50 55 60 Gly Gly Cys Glu Leu

<210> 121 <211> 69 <212> PRT

<213> Artificial Sequence <220> <223> Synthetic R31L <400> 121 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Leu Lys 20 25 30 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 35 40 45 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 50 55 60 Gly Gly Cys Glu 65 Leu <210> 122 <211> 69 <212> PRT <213> Artificial Sequence <220> <223> Synthetic R31N <400> 122 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Asn Lys 20 25 30 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 35 40 45 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 50 55 60

Gly Gly Cys Glu Leu 65 <210> 123 <211> 69

Page 29

735042004740SEQLIST

<212> PRT <213> Artificial Sequence <220> <223> Synthetic L34A <400> 123 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Arg Lys 20 25 30 Lys Ala Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 35 40 45 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 50 55 60 Gly Gly Cys Glu 65 Leu <210> 124 <211> 69 <212> PRT <213> Artificial Sequence <220> <223> Synthetic L34S <400> 124 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Arg Lys 20 25 30 Lys Ser Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 35 40 45 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 50 55 60

Gly Gly Cys Glu Leu 65 <210> 125 <211> 69 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic K28Q/R31A <400> 125

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Gln Arg Gly Ala Lys 20 25 30 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 35 40 45 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 50 55 60 Gly Gly Cys Glu Leu

<210> 126 <211> 69 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic K28Q/R31N

Page 30

735042004740SEQLIST <400> 126

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Gln Arg Gly Asn Lys 20 25 30 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 35 40 45 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 50 55 60 Gly Gly Cys Glu Leu

<210> 127 <211> 69 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic K28Q/R31S <400> 127

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Gln Arg Gly Ser Lys 20 25 30 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 35 40 45 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 50 55 60 Gly Gly Cys Glu Leu

<210> 128 <211> 69 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic K28Q/L34A <400> 128

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Gln Arg Gly Arg Lys 20 25 30 Lys Ala Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 35 40 45 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 50 55 60 Gly Gly Cys Glu Leu

<210> 129 <211> 69 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic K28Q/L34S <400> 129

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Gln Arg Gly Arg Lys

Page 31

735042004740SEQLIST

20 25 30

Lys Ser Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

35 40 45

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

50 55 60

Gly Gly Cys Glu Leu 65 <210> 130 <211> 69 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic R31N/L34A <400> 130

Phe Trp Val Leu Val Val Val Gly 1 5 Leu Val Thr Val Ala Phe Ile Ile 20 Lys Ala Leu Tyr Ile Phe Lys Gln 35 40 Thr Gln Glu Glu Asp Gly Cys Ser 50 55 Gly Gly Cys Glu Leu

Gly Val 10 Leu Ala Cys Tyr Ser 15 Leu Phe 25 Trp Val Lys Arg Gly 30 Asn Lys Pro Phe Met Arg Pro 45 Val Gln Thr Cys Arg Phe Pro 60 Glu Glu Glu Glu

<210> 131 <211> 69 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic R31N/L34S <400> 131

Phe Trp Val Leu Val Val Val Gly 1 5 Leu Val Thr Val Ala Phe Ile Ile 20 Lys Ser Leu Tyr Ile Phe Lys Gln 35 40 Thr Gln Glu Glu Asp Gly Cys Ser 50 55 Gly Gly Cys Glu Leu

<210> 132 <211> 69 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic K28Q/R31N/L34A <400> 132

Phe Trp Val Leu Val Val Val Gly 1 5 Leu Val Thr Val Ala Phe Ile Ile 20 Lys Ala Leu Tyr Ile Phe Lys Gln 35 40 Thr Gln Glu Glu Asp Gly Cys Ser 50 55

Gly Val 10 Leu Ala Cys Tyr Ser 15 Leu Phe 25 Trp Val Gln Arg Gly 30 Asn Lys Pro Phe Met Arg Pro 45 Val Gln Thr Cys Arg Phe Pro 60 Glu Glu Glu Glu

Page 32

735042004740SEQLIST

Gly Gly Cys Glu Leu 65 <210> 133 <211> 69 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic K28Q/R31N/L34S <400> 133

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Gln Arg Gly Asn Lys 20 25 30 Lys Ser Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 35 40 45 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 50 55 60 Gly Gly Cys Glu Leu

<210> 134 <211> 71 <212> PRT

<213> Artificial Sequence <220> <223> Synthetic <220> <223> NN Insertion <400> 134 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Asn Asn Lys Arg Gly 20 25 30 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val 35 40 45 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu 50 55 60 Glu Glu Gly Gly Cys Glu Leu 65 70 <210> 135 <211> 70 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 135 Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser 1 5 10 15 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Arg 20 25 30 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 35 40 45 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

50 55 60

Glu Gly Gly Cys Glu Leu 65 70

Page 33

735042004740SEQLIST <210> 136 <211> 108 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <400> 136

Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn 1 5 10 15 Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu 20 25 30 Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly 35 40 45 Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe 50 55 60 Trp Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 65 70 75 80 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 85 90 95 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 100 105

<210> 137 <211> 26 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> junction region <400> 137

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly 1 5 10 15 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

20 25 <210> 138 <211> 26 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> junction region K14A <400> 138

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Ala Arg Gly 1 5 10 15 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 20 25 <210> 139 <211> 26 <212> PRT <213> Artificial Sequence

Page 34

735042004740SEQLIST <220>

<223> Synthetic <220>

<223> junction region K14H <400> 139

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val His Arg Gly 1 5 10 15

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 20 25 <210> 140 <211> 26 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> junction region K14L <400> 140

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Leu Arg 1 5 10 15 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

20 25 <210> 141 <211> 26 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> junction region K14Q <400> 141

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Gln Arg 1 5 10 15 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln

20 25 <210> 142 <211> 26 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> junction region K14S <400> 142

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Ser Arg 1 5 10 15 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln 20 25

<210> 143

Page 35

735042004740SEQLIST <211> 26 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> junction region R17A <400> 143

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly 1 5 10 15

Ala Lys Lys Leu Leu Tyr Ile Phe Lys Gln 20 25 <210> 144 <211> 26 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> junction region R17H <400> 144

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg 1 5 10 15 His Lys Lys Leu Leu Tyr Ile Phe Lys Gln

20 25 <210> 145 <211> 26 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> junction region R17L <400> 145

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg 1 5 10 15 Leu Lys Lys Leu Leu Tyr Ile Phe Lys Gln

20 25 <210> 146 <211> 26 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> junction region R17N <400> 146

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg 1 5 10 15 Asn Lys Lys Leu Leu Tyr Ile Phe Lys Gln

Page 36

20 735042004740SEQLIST 25 <210> <211> <212> <213> 147 26 PRT Artificial Sequence

<220>

<223> Synthetic <220>

<223> junction region L20A <400> 147

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly 1 5 10 15

Arg Lys Lys Ala Leu Tyr Ile Phe Lys Gln 20 25 <210> 148 <211> 26 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> junction region L20S <400> 148

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly 1 5 10 15

Arg Lys Lys Ser Leu Tyr Ile Phe Lys Gln 20 25 <210> 149 <211> 26 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> junction region K14Q/R17A <400> 149

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Gln Arg Gly 1 5 10 15

Ala Lys Lys Leu Leu Tyr Ile Phe Lys Gln 20 25 <210> 150 <211> 26 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> junction region K14Q/R17N

Page 37

735042004740SEQLIST <400> 150

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Gln Arg 1 5 10 15 Asn Lys Lys Leu Leu Tyr Ile Phe Lys Gln 20 25

<210> 151 <211> 26 <212> PRT <213> Artificial Sequence

<220>

<223> Synthetic <220>

<223> junction region K14Q/R17S <400> 151

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Gln Arg Gly 1 5 10 15

Ser Lys Lys Leu Leu Tyr Ile Phe Lys Gln 20 25 <210> 152 <211> 26 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> junction region K14Q/L20A <400> 152

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Gln Arg Gly 1 5 10 15

Arg Lys Lys Ala Leu Tyr Ile Phe Lys Gln 20 25 <210> 153 <211> 26 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> junction region K14Q/L20S <400> 153

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Gln Arg Gly 1 5 10 15

Arg Lys Lys Ser Leu Tyr Ile Phe Lys Gln 20 25 <210> 154 <211> 26 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic

Page 38

735042004740SEQLIST <220>

<223> junction region R17N/L20A <400> 154

Ser 1 Leu Leu Val Thr 5 Val Ala Phe Ile Ile Phe Trp Val 10 Lys Arg 15 Asn Lys Lys Ala Leu Tyr Ile Phe Lys Gln 20 25

<210> 155 <211> 26 <212> PRT <213> Artificial Sequence

<220>

<223> Synthetic <220>

<223> junction region R17N/L20S <400> 155

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly 1 5 10 15

Asn Lys Lys Ser Leu Tyr Ile Phe Lys Gln 20 25 <210> 156 <211> 26 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> junction region K14Q/R17N/L20A <400> 156

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Gln Arg Gly 1 5 10 15

Asn Lys Lys Ala Leu Tyr Ile Phe Lys Gln 20 25 <210> 157 <211> 26 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> junction region K14Q/R17N/L20S <400> 157

Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Gln Arg Gly 1 5 10 15

Asn Lys Lys Ser Leu Tyr Ile Phe Lys Gln 20 25 <210> 158 <211> 36 <212> DNA

Page 39

735042004740SEQLIST <213> Homo sapiens <220>

<223> spacer (IgG4hinge) <400> 158 gaatctaagt acggaccgcc ctgcccccct tgccct <210> 159 <211> 112 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> CD3 Zeta <400> 159

Arg Val 1 Lys Phe Ser 5 Arg Ser Ala Asp Ala Pro 10 Ala Tyr Lys Gln 15 Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110

<210> 160 <211> 36 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <400> 160

Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys 1 5 10 15 Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg

20 25 30

Pro Val Gln Thr 35 <210> 161 <211> 15 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <400> 161

Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 1 5 10 15

Page 40

735042004740SEQLIST

<210> 162 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 162 Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly 1 5 10 15 <210> 163 <211> 15 <212> PRT <213> Artificial Sequence

<220>

<223> Synthetic <400> 163

Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Arg Lys Lys 1 5 10 15

<210> 164 <211> 15 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <400> 164

Phe Ile Ile Phe Trp Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr 1 5 10 15

<210> 165 <211> 15 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <400> 165

Phe Trp Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys 1 5 10 15

<210> 166 <211> 15 <212> PRT

<213> Artificial Sequence <220> <223> Synthetic <400> 166 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 1 5 10 15

<210> 167 <211> 15 <212> PRT

Page 41

735042004740SEQLIST <213> Artificial Sequence <220>

<223> Synthetic <400> 167

Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 1 5 10 15 <210> 168 <211> 15 <212> PRT <213> Artificial Sequence

<220>

<223> Synthetic <400> 168

Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 1 5 10 15 <210> 169 <211> 15 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31H <400> 169

Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly His Lys Lys 1 5 10 15

<210> 170 <211> 15 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31H <400> 170

Phe Ile Ile Phe Trp Val Lys Arg Gly His Lys Lys Leu Leu Tyr 1 5 10 15

<210> 171 <211> 15 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31H <400> 171

Phe Trp Val Lys Arg Gly His Lys Lys Leu Leu Tyr Ile Phe Lys 1 5 10 15

<210> 172 <211> 15 <212> PRT

<213> Artificial Sequence <220> Page 42

735042004740SEQLIST <223> Synthetic peptide R31H <400> 172

Lys Arg Gly His Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 1 5 10 15 <210> 173 <211> 15 <212> PRT <213> Artificial Sequence

<220>

<223> Synthetic peptide R31H <400> 173

His Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 1 5 10 15

<210> 174 <211> 15 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31N <400> 174

Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Asn Lys Lys 1 5 10 15

<210> 175 <211> 15 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31N <400> 175

Phe Ile Ile Phe Trp Val Lys Arg Gly Asn Lys Lys Leu Leu Tyr 1 5 10 15

<210> 176 <211> 15 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide R31N <400> 176

Phe Trp Val Lys Arg Gly Asn Lys Lys Leu Leu Tyr Ile Phe Lys 1 5 10 15

<210> <211> <212> <213> 177 15 PRT Artificial Sequence <220> <223> Synthetic peptide R31N <400> 177

Page 43

735042004740SEQLIST

Lys Arg Gly Asn Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe 1 5 10 15 <210> 178 <211> 15 <212> PRT <213> Artificial Sequence

<220>

<223> Synthetic peptide R31N <400> 178

Asn Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 1 5 10 15 <210> 179 <211> 15 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide L34A <400> 179

Phe Ile Ile Phe Trp Val Lys Arg Gly Arg Lys Lys Ala Leu Tyr 1 5 10 15

<210> 180 <211> 15 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide L34A <400> 180

Phe Trp Val Lys Arg Gly Arg Lys Lys Ala Leu Tyr Ile Phe Lys 1 5 10 15 <210> 181 <211> 15 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic peptide L34A <400> 181

Lys Arg Gly Arg Lys Lys Ala Leu Tyr Ile Phe Lys Gln Pro Phe 1 5 10 15 <210> 182 <211> 15 <212> PRT

<213> Artificial Sequence <220> <223> Synthetic peptide L34A <400> 182 Arg Lys Lys Ala Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 1 5 10 15

Page 44

735042004740SEQLIST <210> 183 <211> 69 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic R31S <400> 183

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Lys Arg Gly Ser Lys 20 25 30 Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 35 40 45 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu 50 55 60 Gly Gly Cys Glu Leu

<210> 184 <211> 26 <212> PRT <213> Artificial Sequence <220>

<223> Synthetic <220>

<223> junction region R31S <400> 184

Ser 1 Leu Leu Val Thr 5 Val Ala Phe Ile Ile Phe Trp Val 10 Lys Arg Gly 15 Ser Lys Lys Leu Leu Tyr Ile Phe Lys Gln 20 25

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